Liver organoid, uses thereof and culture method for obtaining them

ABSTRACT

The invention relates to a liver organoid, uses thereof and method for obtaining them.

RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. §371 ofinternational application PCT/IB2011/002167, filed Jul. 29, 2011, whichwas published under PCT Article 21(2) in English, and claims the benefitunder 35 U.S.C. §119(e) and the right of priority to U.S. ProvisionalApplication Ser. No. 61/368,736, filed on Jul. 29, 2010 and U.S.Provisional Application Ser. No. 61/520,569, filed on Jun. 10, 2011, andwhich also claims priority to European Application Serial No.10171265.1, filed on Jul. 29, 2010, the entire contents of each of whichare incorporated by reference herein in their entireties.

FIELD OF THE INVENTION

The invention relates to a liver organoid, uses thereof and method forobtaining them.

BACKGROUND OF THE INVENTION

The basic architectural unit of the liver is the liver lobule. Eachlobule consists of plates of hepatocytes lined by sinusoidal capillariesthat radiate toward a central efferent vein. Liver lobules are roughlyhexagonal with each of six corners demarcated by the presence of aportal triad (portal vein, bile duct, and hepatic artery). Althoughhepatocytes are the major parenchymal cell type of the liver theyfunction in concert with cholangiocytes (biliary epithelial cells),endothelial cells, sinusoidal endothelial cells, Kupffer cells, naturalkiller cells and hepatic stellate cells. This complex architecture iscrucial for hepatic function.

The existence of liver stem cells remains controversial. On one hand,tissue maintenance in the liver and liver regeneration upon certaintypes of injury, are not driven by stem cells but rather by division ofthe mature cells (hepatocytes or cholangyocytes). However, liver injurymodels in which hepatocyte proliferation has been inhibited alsodemonstrated the ability of the organ to regenerate in response todamage. This suggests that the liver can be considered as an organ withfacultative stem cells.

So far, liver cultures derived from hepatocytes, or by differentiationof Embryonic Stem cells (ES) or induced Pluripotent Stem Cells, do notexpand and self renew for longer periods.

Recently, in the small intestine, the gene Lgr5 was identified which isspecifically expressed in cycling Crypt Base Columnar (CBC) cells, whichare small cells that are interspersed between the Paneth cells (Barkeret al., 2007. Nature 449: 1003-1007). Using a mouse in which aGFP/tamoxifen-inducible Cre recombinase cassette was integrated into theLgr5 locus, it was shown by lineage tracing that the Lgr5⁺ CBC cellsconstitute multipotent stem cells which generate all cell types of theepithelium even when assessed 14 months after Cre induction.

It was recently discovered that also Lgr6, besides Lgr5, but not Lgr4,is a unique marker for adult stem cells. While Lgr5 is expressed in stemcells of brain, kidney, liver, retina, stomach, intestine, pancreas,breast, hair follicle, ovary, adrenal medulla, and skin, Lgr6 isexpressed in stem cells of brain, lung, breast, hair follicle, and skin.

Here we have developed a method to culture adult liver progenitors andto obtain a liver organoid that shows longer-lived maintenance, are ableto differentiate to both, hepatocyte and cholangiocyte lineages andpreserve the basic physiology of isolated liver fragments.

SUMMARY OF THE INVENTION

The invention provides a method for obtaining a liver organoid, whereinthe method comprises culturing cells in a first “expansion” culturemedium (also referred to herein as EM), preferably followed by culturingthe cells in a second “differentiation” culture medium (also referred toherein as DM).

The liver organoid may be obtained by culturing a single Lgr5+ stemcell, a population of cells comprising at least one Lgr5+ stem cell,and/or a liver fragment. Herein, where “cells in/of the culture medium”are referred to, the meaning includes a single Lgr5+ stem cell, apopulation of cells comprising at least one Lgr5+ stem cell, and/or aliver fragment.

In one embodiment, the expansion medium comprises EGF, a Wnt agonist,FGF, and Nicotinamide. Preferably, the Wnt agonist is R-spondin 1 and sothe expansion medium is referred to as “ERFNic”. A particularlypreferred expansion medium additionally comprises HGF and is referred toas “ERFHNic”.

In one embodiment, the differentiation medium comprises EGF, a TGF-betainhibitor, FGF and a Notch inhibitor. In one embodiment, the TGF-betainhibitor is A83-01 and/or the Notch inhibitor is DAPT. Thisdifferentiation medium is referred to herein as “EAFD” and is apreferred differentiation medium of the invention. FGF may optionally bereplaced by HGF or alternatively both FGF and HGF may be present orabsent in the differentiation medium. Dexamethasone may also be added.

In a preferred embodiment, the cells may initially be cultured in anexpansion medium that additionally contains Wnt and Noggin, for examplean “ENRW” medium containing EGF, Noggin, R-spondin and Wnt, andoptionally FGF, HGF and Nicotinamide. The inventors have found that thismedium is optimum for stimulating initial expansion of cells for thefirst few days. Therefore, this first expansion medium is sometimesreferred to herein as EM1. In some embodiments, the Wnt and Noggin areremoved after approximately 1 day, 2 days, 3 days, 4 days, 5 days, 6days, 7 days or more, for example 2 week, 1 month, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12 months or more, 14 months or more. The cells may then beexpanded in an expansion medium of the invention, as described abovethat does not contain Wnt or Noggin. This second expansion medium issometimes referred to herein as EM2. In some embodiments, the cells arecultured in EM2 for approximately 1 day, 2 days, 3 days, 4 days, 5 days,6 days, 7 days or a longer time period, such as 3, 4, 5, 10, 20 or moreweeks. The culture medium may then be changed to an optimiseddifferentiation medium, as described above, that contains a TGF-betainhibitor and a Notch inhibitor. Typically, the differentiation mediumdoes not contain a Wnt agonist, R-spondin or Nicotinamide. Thisencourages the differentiation of the cells towards mature hepatocytesand cholangyocytes. These cells are suitable for transplantation intohumans or animals.

The invention also provides a liver organoid. The present applicationdescribes the first time that liver organoids have been grown ex vivo.

DESCRIPTION OF THE INVENTION

In a first aspect, the invention provides a method for obtaining and/orculturing a liver fragment or a liver organoid, wherein said methodcomprises:

culturing epithelial stem cells, and/or isolated tissue fragmentscomprising said epithelial stem cells in contact with an extracellularmatrix in the presence of a medium, the medium comprising a basal mediumfor animal or human cells to which is added: Epidermal Growth Factor, anFGF able to bind to FGFR2 or FGFR4, preferably FGF10 as a mitogenicgrowth factor, Nicotinamide, and preferably, a Wnt agonist, preferablyR-spondin 1, R-spondin 2, R-spondin 3 or R-spondin 4 and/or Wnt-3a.Preferably, HGF is also added.

For example, in one embodiment, the invention provides a method forobtaining and/or culturing a liver fragment or a liver organoid, whereinsaid method comprises:

culturing epithelial stem cells, and/or isolated tissue fragmentscomprising said epithelial stem cells in contact with an extracellularmatrix in the presence of a medium, the medium comprising a basal mediumfor animal or human cells to which is added a BMP inhibitor, a Wntagonist, Epidermal Growth Factor, a FGF able to bind to FGFR2 or FGFR4,preferably FGF10 and HGF as mitogenic growth factors, gastrin,Nicotinamide, B27, N2 and N-Acetylcysteine.

It has surprisingly been found by the present inventors that a method ofthe invention allows culturing of epithelial stem cells, and/or isolatedfragments from the liver comprising said stem cells, while preservingthe presence of stem cells that retain an undifferentiated phenotype andself-maintenance capabilities. Even more surprising was the observationthat a method of the invention allows the outgrowth of a single,isolated epithelial stem cell into a liver organoid in the absence of astem cell niche.

Stem cells are found in many organs of adult humans and mice. Althoughthere may be great variation in the exact characteristics of adult stemcells in individual tissues, adult stem cells share at least thefollowing characteristics: they retain an undifferentiated phenotype;their offspring can differentiate towards all lineages present in thepertinent tissue; they retain self-maintenance capabilities throughoutlife; and they are able to regenerate the pertinent tissue after injury.Stem cells reside in a specialized location, the stem cell niche, whichsupplies the appropriate cell-cell contacts and signals for maintenanceof said stem cell population.

Epithelial stem cells are able to form the distinct cell types of whichthe epithelium is composed. Some epithelia, such as skin or intestine,show rapid cell turnover, indicating that the residing stem cells mustbe continuously proliferating. Other epithelia, such as the liver orpancreas, show a very slow turnover under normal conditions.

The isolation of liver epithelial stem cells may be carried out by anumber of different protocols. Although the inventors do not wish to bebound by any particular theory, it is hypothesised herein that apopulation of stem cells exists within the liver that upon tissueinjury, is responsible for liver regeneration. It is thought that thecell population responsible for this injury-responsive regenerationexpresses the marker Lgr5 when activated.

By injecting Lgr5-LacZ knock-in mice with the hepatotoxic compound CC14to simulate liver damage, the inventors have demonstrated for the firsttime that liver damage induces Lgr5 expression in the liver at sites ofactive regeneration (see example 4). The inventors have also shown thatinjection of the Wnt agonist R-spondin causes upregulation of Wntsignalling expression in the liver ducts. Although the inventors do notwish to be bound by any particular theory, it is hypothesised hereinthat regenerative stem cells/progenitors may be activated by Wntsignalling after liver injury. The Lgr5 positive cells may then beisolated from the liver/liver fragment, if required, as describedherein. Thus the invention provides a method for obtaining/isolatingLgr5+ cells from the liver comprising inducing liver injury or bystimulating Wnt signalling, for example with R-spondin. This is usefulbecause in some embodiments Lgr5+ cells are the starting point forobtaining liver organoids in vitro (although the use of a culture mediumof the invention allows generation of a Lgr5+ cell by adding a Wntagonist such as R-spondin and so it is not essential to obtain an Lgr5+cell from the liver in order to practice the present invention). Lgr5+cells obtained by this method are also provided. In some embodiments,such cells preferably do not express markers of stellate cells, forexample SMA. Instead, such cells preferably express one or more liverprogenitor markers or stem cell markers such as Sox9. Preferably, theexpression of one or more liver progenitor markers or stem cell markerssuch as Sox9 is upregulated in said cells compared to adult liver.

The invention also provides a method for regenerating the livercomprising stimulating Wnt signalling. Such a method may be useful inthe treatment of liver disorders. The induction of Lgr5 expression inliver cells by injury or by stimulating Wnt signalling may be carriedout in vivo, ex vivo in an isolated liver, or in vitro in a liverfragment or population of liver cells. In embodiments in which theinduction of Lgr5 expression is carried out ex vivo or in vitro, theLgr5 positive cells may be administered to a patient in need thereof byany suitable means, for example, by injection or by implantation. Insome embodiments, implanted cells are contained in biocompatible matrix.In some embodiments, the cells are expanded ex vivo prior to being usedin therapy.

In a preferred method of the invention, said epithelial stem cells areisolated from a liver fragment or a liver biliary duct, more preferablyfrom biliary duct tissue.

Methods for the isolation of bile duct tissue are known to those ofskill in the art. For example, biliary duct may be isolated from a liveras described in the examples enclosed herein. Briefly, an adult livertissue may be washed in a cold (4-10° C.) culture medium, preferablyAdvanced-DMEM/F12 (Invitrogen) and then, the tissue can be chopped intopieces of around 5 mm and further washed with cold dissociation buffer(collagenase, dispase, FBS in DMEM media). The tissue fragments arepreferably incubated with the dissociation buffer for about 2 h at about37° C. Then, the tissue fragments can be vigorously suspended in 10 mlof cold (4-10° C.) isolation buffer with a 10 ml pipette. The firstsupernatant containing dead cells is preferably discarded and thesediment preferably suspended with dissociation buffer (e.g. 10-15 ml).After further vigorous suspension of the tissue fragments thesupernatant is enriched in biliary ducts. A suspension containingbiliary ducts can in this way be obtained and biliary ducts arecollected under the microscope and retained in cold media (DMEM+5-10%FBS). This procedure may be repeated until at least 10-20 biliaryducts/well are collected. Then, the isolated biliary ducts may beprecipitated. Isolated bile ducts are preferably seeded in 50 ul ofMATRIGEL™ at an approximate ratio of 20 biliary ducts/well.

In contrast to mature hepatocytes, which grow to confluence for a shortperiod of time, before dying, liver epithelial stem cells isolatedaccording to the invention are self-renewing and grow indefinitely. Ithas been found that the self-renewing population of cells are thosewhich are capable of expressing Lgr5 on their surface. Lgr5 negativecells do not self-renew. The term “self-renewing” should be understoodto represent the capacity of a cell to reproduce itself whilstmaintaining the original proliferation and differentiation properties ofcells of the invention. Such cells proliferate by dividing to formclones, which further divide into clones and therefore expand the sizeof the cell population without the need for external intervention,without evolving into cells with a more restricted differentiationpotential.

A preferred method is based on the fact that liver stem cells accordingto the invention express Lgr5 and/or Lgr6 on their surface; theseproteins belong to the large G protein-coupled receptor (GPCR)superfamily (see, for example, WO2009/022907, the contents of which areincorporated herein in their entirety). The Lgr subfamily is unique incarrying a large leucine-rich ectodomain important for ligand binding. Apreferred method therefore comprises preparing a cell suspension fromsaid epithelial tissue as described above, contacting said cellsuspension with an Lgr5 and/or 6 binding compound (such as an antibody),isolating the Lgr5 and/or 6 binding compound, and isolating the stemcells from said binding compound.

It is preferred that a single cell suspension comprising the epithelialstem cells is mechanically generated from the isolated biliary duct.Small organoid fragments generated in this way by mechanical disruptionare preferably split at a ratio of approximately 1:6. If necessary, suchfragments can be incubated for a short time (only 2 or 3 minutes) intrypsin at a dilution of approximately 1:2. It has been found that atthis stage epithelial stem cells treated with trypsin yielded rather lowsurvival rates: if the cells are split into individual cells, then onlythose expressing Lgr5 survive. This fraction is rather small(approximately 12% of the total cell population).

Preferred Lgr5 and/or 6 binding compounds comprise antibodies, such asmonoclonal antibodies that specifically recognize and bind to theextracellular domain of either Lgr5 or Lgr6, such as monoclonalantibodies including mouse and rat monoclonal antibodies (see, forexample, WO2010/016766, the contents of which are incorporated herein intheir entirety). Using such an antibody, Lgr5 and/or Lgr6-expressingstem cells can be isolated, for example with the aid of magnetic beadsor through fluorescence-activated cell sorting, as is clear to a skilledperson. Using a method of the invention, it is possible to isolate onesingle Lgr5 and/or Lgr6 expressing cell and to apply a method of theinvention to it. A liver organoid may therefore be derived from onesingle cell.

Stem Cell Niche

Isolated stem cells are preferably cultured in a microenvironment thatmimics at least in part a cellular niche in which said stem cellsnaturally reside. This cellular niche may be mimicked by culturing saidstem cells in the presence of biomaterials, such as matrices, scaffolds,and culture substrates that represent key regulatory signals controllingstem cell fate. Such biomaterials comprise natural, semi-synthetic andsynthetic biomaterials, and/or mixtures thereof. A scaffold provides atwo-dimensional or three dimensional network. Suitable syntheticmaterials for such a scaffold comprise polymers selected from poroussolids, nanofibers, and hydrogels such as, for example, peptidesincluding self-assembling peptides, hydrogels composed of polyethyleneglycol phosphate, polyethylene glycol fumarate, polyacrylamide,polyhydroxyethyl methacrylate, polycellulose acetate, and/or co-polymersthereof (see, for example, Saha et al., 2007. Curr Opin Chem Biol.11(4): 381-387; Saha et al., 2008. Biophysical Journal 95: 4426-4438;Little et al., 2008. Chem. Rev 108, 1787-1796). As is known to a skilledperson, the mechanical properties such as, for example, the elasticityof the scaffold influences proliferation, differentiation and migrationof stem cells. A preferred scaffold comprises biodegradable (co)polymersthat are replaced by natural occurring components after transplantationin a subject, for example to promote tissue regeneration and/or woundhealing. It is furthermore preferred that said scaffold does notsubstantially induce an immunogenic response after transplantation in asubject. Said scaffold is supplemented with natural, semi-synthetic orsynthetic ligands, which provide the signals that are required forproliferation and/or differentiation, and/or migration of stem cells. Ina preferred embodiment, said ligands comprise defined amino acidfragments. Examples of said synthetic polymers comprise Pluronic® F127block copolymer surfactant (BASF), and Ethisorb® (Johnson and Johnson).

A cellular niche is in part determined by the stem cells and surroundingcells, and the extracellular matrix (ECM) that is produced by the cellsin said niche. In a preferred method of the invention, isolated liverfragments or isolated biliary duct or epithelial stem cells are attachedto an ECM. ECM is composed of a variety of polysaccharides, water,elastin, and glycoproteins, wherein the glycoproteins comprise collagen,entactin (nidogen), fibronectin, and laminin. ECM is secreted byconnective tissue cells. Different types of ECM are known, comprisingdifferent compositions including different types of glycoproteins and/ordifferent combination of glycoproteins. Said ECM can be provided byculturing ECM-producing cells, such as for example fibroblast cells, ina receptacle, prior to the removal of these cells and the addition ofisolated liver fragments or isolated biliary duct or isolated epithelialstem cells. Examples of extracellular matrix-producing cells arechondrocytes, producing mainly collagen and proteoglycans, fibroblastcells, producing mainly type IV collagen, laminin, interstitialprocollagens, and fibronectin, and colonic myofibroblasts producingmainly collagens (type I, III, and V), chondroitin sulfate proteoglycan,hyaluronic acid, fibronectin, and tenascin-C. Alternatively, said ECM iscommercially provided. Examples of commercially available extracellularmatrices are extracellular matrix proteins (Invitrogen) and basementmembrane preparations from Engelbreth-Holm-Swarm (EHS) mouse sarcomacells (e.g. MATRIGEL™ (BD Biosciences)). A synthetic extracellularmatrix material, such as ProNectin (Sigma Z378666) may be used. Mixturesof extracellular matrix materials may be used, if desired. The use of anECM for culturing stem cells enhanced long-term survival of the stemcells and the continued presence of undifferentiated stem cells. In theabsence of an ECM, stem cell cultures could not be cultured for longerperiods and no continued presence of undifferentiated stem cells wasobserved. In addition, the presence of an ECM allowed culturing ofthree-dimensional tissue organoids, which could not be cultured in theabsence of an ECM. The extracellular matrix material will normally becoated onto a cell culture vessel, but may (in addition oralternatively) be supplied in solution. A fibronectin solution of about1 mg/ml may be used to coat a cell culture vessel, or between about 1μg/cm² to about 250 μg/cm², or at about 1 μg/cm² to about 150 μg/cm². Insome embodiments, a cell culture vessel is coated with fibronectin atbetween 8 μg/cm² and 125 μg/cm².

A preferred ECM for use in a method of the invention comprises at leasttwo distinct glycoproteins, such as two different types of collagen or acollagen and laminin. The ECM can be a synthetic hydrogel extracellularmatrix or a naturally occurring ECM. A further preferred ECM is providedby MATRIGEL™ (BD Biosciences), which comprises laminin, entactin, andcollagen IV.

The compositions of the invention may comprise serum, or may beserum-free and/or serum-replacement free, as described elsewhere herein.Culture media and cell preparations are preferably GMP processes in linewith standards required by the FDA for biologics products and to ensureproduct consistency.

Culture Media

A cell culture medium that is used in a method of the inventioncomprises any suitable cell culture medium, subject to the limitationsprovided herein. Cell culture media typically contain a large number ofingredients, which are necessary to support maintenance of the culturedcells. Suitable combinations of ingredients can readily be formulated bythe skilled person, taking into account the following disclosure. Aculture medium according to the invention will generally be a nutrientsolution comprising standard cell culture ingredients, such as aminoacids, vitamins, inorganic salts, a carbon energy source, and a buffer,as described in more detail in the literature and below.

A culture medium of the invention will normally be formulated indeionized, distilled water. A culture medium of the invention willtypically be sterilized prior to use to prevent contamination, e.g. byultraviolet light, heating, irradiation or filtration. The culturemedium may be frozen (e.g. at −20° C. or −80° C.) for storage ortransport. The medium may contain one or more antibiotics to preventcontamination. The medium may have an endotoxin content of less that 0.1endotoxin units per ml, or may have an endotoxin content less than 0.05endotoxin units per ml. Methods for determining the endotoxin content ofculture media are known in the art.

A preferred cell culture medium is a defined synthetic medium that isbuffered at a pH of 7.4 (preferably with a pH 7.2-7.6 or at least 7.2and not higher than 7.6) with a carbonate-based buffer, while the cellsare cultured in an atmosphere comprising between 5% and 10% CO2, or atleast 5% and not more than 10% CO2, preferably 5% CO2.

The skilled person will understand from common general knowledge thetypes of culture media that might be used for as the basal medium in thecell culture mediums of the invention. Potentially suitable cell culturemedia are available commercially, and include, but are not limited to,Dulbecco's Modified Eagle Media (DMEM), Minimal Essential Medium (MEM),Knockout-DMEM (KO-DMEM), Glasgow Minimal Essential Medium (G-MEM), BasalMedium Eagle (BME), DMEM/Ham's F12, Advanced DMEM/Ham's F12, Iscove'sModified Dulbecco's Media and Minimal Essential Media (MEM), Ham's F-10,Ham's F-12, Medium 199, and RPMI 1640 Media.

A preferred cell culture medium is selected from DMEM/F12 and RPMI 1640supplemented with glutamine, insulin, Penicillin/streptomycin andtransferrin. In a further preferred embodiment, Advanced DMEM/F12 orAdvanced RPMI is used, which is optimized for serum free culture andalready includes insulin. In this case, said Advanced DMEM/F12 orAdvanced RPMI medium is preferably supplemented with glutamine andPenicillin/streptomycin.

In some embodiments, the basal medium comprises Advanced DMEM F12,hepes, penicillin/streptomycin, Glutamin, NAcetyl Cystein, B27, N2 andGastrin. In some embodiments, culture is initiated with a basal mediumcomprising N2 and Gastrin and penicillin/streptomycin but these arelater withdrawn. For example, in some embodiments, N2 and Gastrin andpenicillin/streptomycin are present in an EM1 medium of the inventionbut not in an EM2 or DM. For example, in some embodiments, N2 andGastrin and penicillin/streptomycin are present in an EM1 and EM2 mediumof the invention but not in a DM. In particularly preferred embodiments,the basal medium is Advanced DMEM/F12 or a DMEM variant supplementedwith penicillin/streptomycin, N2, B27, glutamine and gastrin.

In preferred embodiments, the basal medium comprises Gastrin. In someembodiments, the basal medium also comprises NAc and/or B27.

It is furthermore preferred that said cell culture medium issupplemented with a purified, natural, semi-synthetic and/or syntheticgrowth factor and does not comprise an undefined component such as fetalbovine serum or fetal calf serum. Various different serum replacementformulations are commercially available and are known to the skilledperson. Where a serum replacement is used, it may be used at betweenabout 1% and about 30% by volume of the medium, according toconventional techniques.

Expansion Medium (EM2):

In one aspect of the present invention there is provided a cell culturemedium which comprises or consists of a basal medium for animal or humancells to which is added: Epidermal Growth Factor, an FGF able to bind toFGFR2 or FGFR4, preferably FGF10 as a mitogenic growth factor,Nicotinamide, and preferably, a Wnt agonist, preferably R-spondin 1.This medium is referred to as EM2.

Preferably, the Wnt agonist is R-spondin 1. A medium comprising EGF,R-spondin 1, FGF and Nicotinamide is referred to herein as ERFNic.

In some embodiments, the EM2 medium does not comprise noggin, and morepreferably does not comprise a BMP-inhibitor. In some embodiments, theEM2 medium does not comprise Wnt, for example Wnt-3a.

In some embodiments, HGF is present in addition to FGF. A preferredmedium comprising HGF in addition to FGF is ERFHNic (EGF+R-spondin(preferably R-spondin 1)+FGF (preferably FGF10)+HGF+Nicotinamide). Theinventors have found that the ERFHNic medium is the optimal medium forlong-term expansion of cells. In the absence of HGF, cells did notremain viable in culture for longer than three months. Further, in theabsence of HGF, after 10 passages, cells showed a growth disadvantagecompared to cells cultured in the presence of HGF as evidenced by alower proliferation ratio. In particular, after 15 passages, the cellswere not growing organoids at the same speed ratio in the absence of HGFas in the presence of HGF. Thus, HGF was found to be essential formaintaining a good proliferation rate during long-term culture. Thus theinvention provides the use of an ERFHNic medium of the invention forculturing cells for at least 2 weeks, at least 1 month, at least 2months, more preferably at least 3, at least 4, at least 5, at least 6,at least 7, at least 8, at least 9, at least 10, at least 15, at least20, at least 24, at least 25, at least 30 or more months, for example 3or more years. In practice, some embodiments of the invention comprisethe use of E2 for around 20-30 passages of the cells. For example, thecells may be split 20-30 times, generally once a week. Preferably thecells will expand at a rate of about 4 fold per week or two populationdoublings a week. The invention further provides the use of an ERFHNicmedium of the invention for culturing cells for at least 10 passages,for example, at least 11, at least 12, at least 15, at least 20, atleast 25, at least 30, at least 40, at least 50, at least 60 passages orfor between 15-35 passages, for example approximately 20-30 passages. Insome embodiments, a TGF-beta inhibitor such as A83-01 is additionallypresent in the EM2 medium. This is particularly useful when human cellsor organoids are being cultured. In some embodiments, the A83-01 ispresent at a concentration of between 400-600 nM, for example 450-550nM, 470-530 nM or approximately 500 nM. In embodiments in which aTGF-beta inhibitor is present in EM2, a Notch inhibitor is preferablynot present.

Expansion Medium (EM1):

In one aspect, the invention provides a cell culture medium comprisingor consisting of a basal medium for animal or human cells to which isadded EGF, a BMP inhibitor, R-spondin and Wnt. Preferably, the BMPinhibitor is Noggin and the EM1 medium is termed “ENRW” (EGF, Noggin,R-spondin and Wnt). This medium is referred to as EM1. The inventorshave found that a medium containing Wnt and Noggin is ideal forstimulating initial expansion of cells. Thus, in some embodiments, theEM1 medium is used for just 1 passage or 1 week but it is also envisaegdthat EM1 medium can be used for around a year because it is not farmfulfor the cells. Thus, in some embodiments, an EM1 medium is used forculturing cells from day 0 to day 10, for example from days 0-7, days0-6, days 0-5, days 0-4, days 0-3, days 0-2, days 0-1, wherein day 0 isthe day that the cells are isolated from their tissue of origin and day1 is the subsequent day or is used for 1 or more weeks for example 2, 3,4 or more weeks or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more months.In some embodiments, the EM1 medium is used only for the first day orfirst two days of culture. In some embodiments, EM1 medium is used for 1or more passages, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20,25, 30 or more passages, for example, 20-30 passages. In someembodiments, the EM1 medium is used subsequent to a freezing step or anyother transportation step involving a medium or temperature change thatdoes not combine with optimal growth.

The EM1 medium is supplemented with one or more of the compoundsselected from the group consisting of FGF, HGF, Nicotinamide, gastrin,B27, N-acetylcystein and N2. In the case of starting the cultures from afrozen stock or from a single cell, the EM1 media is preferablysupplemented with a ROCK inhibitor. Y27632 is the preferred ROCKinhibitor for use in the invention.

Thus, in one embodiment there is provided a cell culture medium whichcomprises or consists of a basal medium for animal or human cells towhich is added: Epidermal Growth Factor, an FGF able to bind to FGFR2 orFGFR4, preferably FGF10 and HGF as mitogenic growth factors,

gastrin, Nicotinamide, B27, N2 and N-Acetylcysteine, and preferably;

a BMP inhibitor, preferably Noggin; and

a Wnt agonist, preferably R-spondin 1 and/or Wnt-3a.

B27 (Invitrogen), N-Acetylcysteine (Sigma) and N2 (Invitrogen), Gastrin(Sigma) and Nicotinamide (Sigma) are also added to the medium definedabove and are believed to stimulate proliferation of the cells. In thecontext of the invention, Nicotinamide is also referred to herein as“Nic”.

‘N2 Supplement’ is available from Invitrogen, Carlsbad, Calif.;www.invitrogen.com; catalog no. 17502-048; and from PAA LaboratoriesGmbH, Pasching, Austria; www.paa.com; catalog no. F005-004; Bottenstein& Sato, PNAS, 76(1):514-517, 1979. N2 Supplement is supplied by PAALaboratories GmbH as a 100× liquid concentrate, containing 500 μg/mlhuman transferrin, 500 μg/ml bovine insulin, 0.63 μg/ml progesterone,1611 μg/ml putrescine, and 0.52 μg/ml sodium selenite. N2 Supplement maybe added to a culture medium as a concentrate or diluted before additionto a culture medium. It may be used at a 1× final concentration or atother final concentrations. Use of N2 Supplement is a convenient way toincorporate transferrin, insulin, progesterone, putrescine and sodiumselenite into a culture medium of the invention.

‘B27 Supplement’ (available from Invitrogen, Carlsbad, Calif.;www.invitrogen.com; currently catalog no. 17504-044; and from PAALaboratories GmbH, Pasching, Austria; www.paa.com; catalog no. F01-002;Brewer et al., J Neurosci Res., 35(5):567-76, 1993) may be used toformulate a culture medium that comprises biotin, cholesterol, linoleicacid, linolenic acid, progesterone, putrescine, retinol, retinylacetate, sodium selenite, tri-iodothyronine (T3), DL-alpha tocopherol(vitamin E), albumin, insulin and transferrin. B27 Supplement issupplied by PAA Laboratories GmbH as a liquid 50× concentrate,containing amongst other ingredients biotin, cholesterol, linoleic acid,linolenic acid, progesterone, putrescine, retinol, retinyl acetate,sodium selenite, tri-iodothyronine (T3), DL-alpha tocopherol (vitaminE), albumin, insulin and transferrin. Of these ingredients at leastlinolenic acid, retinol, retinyl acetate and tri-iodothyronine (T3) arenuclear hormone receptor agonists. B27 Supplement may be added to aculture medium as a concentrate or diluted before addition to a culturemedium. It may be used at a 1× final concentration or at other finalconcentrations. Use of B27 Supplement is a convenient way to incorporatebiotin, cholesterol, linoleic acid, linolenic acid, progesterone,putrescine, retinol, retinyl acetate, sodium selenite, tri-iodothyronine(T3), DL-alpha tocopherol (vitamin E), albumin, insulin and transferrininto a culture medium of the invention.

BMP Inhibitors

A component that is preferably added to the basal culture medium is aBMP inhibitor. BMPs bind as a dimeric ligand to a receptor complexconsisting of two different receptor serine/threonine kinases, type Iand type II receptors. The type II receptor phosphorylates the type Ireceptor, resulting in the activation of this receptor kinase. The typeI receptor subsequently phosphorylates specific receptor substrates(SMAD), resulting in a signal transduction pathway leading totranscriptional activity.

A BMP inhibitor is defined as an agent that binds to a BMP molecule toform a complex wherein the BMP activity is neutralized, for example bypreventing or inhibiting the binding of the BMP molecule to a BMPreceptor. Alternatively, said inhibitor is an agent that acts as anantagonist or reverse agonist. This type of inhibitor binds with a BMPreceptor and prevents binding of a BMP to said receptor. An example of alatter agent is an antibody that binds a BMP receptor and preventsbinding of BMP to the antibody-bound receptor.

A BMP inhibitor may be added to the media in an amount effective toinhibit a BMP-dependent activity in a cell to at most 90%, morepreferred at most 80%, more preferred at most 70%, more preferred atmost 50%, more preferred at most 30%, more preferred at most 10%, morepreferred 0%, relative to a level of a BMP activity in the absence ofsaid inhibitor, as assessed in the same cell type. As is known to askilled person, a BMP activity can be determined by measuring thetranscriptional activity of BMP, for example as exemplified inZilberberg et al., 2007. BMC Cell Biol. 8:41.

Several classes of natural BMP-binding proteins are known, includingNoggin (Peprotech), Chordin and chordin-like proteins (R&D systems)comprising chordin domains, Follistatin and follistatin-related proteins(R&D systems) comprising a follistatin domain, DAN and DAN-like proteins(R&D systems) comprising a DAN cysteine-knot domain, sclerostin/SOST(R&D systems), decorin (R&D systems), and alpha-2 macroglobulin (R&Dsystems).

A preferred BMP inhibitor for use in a method of the invention isselected from Noggin, DAN, and DAN-like proteins including Cerberus andGremlin (R&D systems). These diffusible proteins are able to bind a BMPligand with varying degrees of affinity and inhibit their access tosignalling receptors. The addition of any of these BMP inhibitors to thebasal culture medium prevents the loss of stem cells.

A preferred BMP inhibitor is Noggin. In the context of a culture mediumof the invention, Noggin is also referred to herein as “N”. Noggin ispreferably added to the basal culture medium at a concentration of atleast 10 ng/ml, more preferred at least 20 ng/ml, more preferred atleast 50 ng/ml, more preferred at least 100 ng/ml. A still morepreferred concentration is approximately 100 ng/ml or exactly 100 ng/ml.During culturing of stem cells, said BMP inhibitor may be added to theculture medium when required, for example, daily or every other day. TheBMP inhibitor is preferably added to the culture medium every secondday. The culture medium may be refreshed when required, for example,daily or every other day, although is preferably refreshed every fourthday.

Wnt Agonists

A further component that may be added to the basal culture medium is aWnt agonist. In the context of a culture medium of the invention, theWnt agonist is also referred to herein as “W”. The Wnt signallingpathway is defined by a series of events that occur when a Wnt proteinbinds to a cell-surface receptor of a Frizzled receptor family member.This results in the activation of Dishevelled family proteins whichinhibit a complex of proteins that includes axin, GSK-3, and the proteinAPC to degrade intracellular β-catenin. The resulting enriched nuclearβ-catenin enhances transcription by TCF/LEF family transcriptionfactors.

A Wnt agonist is defined as an agent that activates TCF/LEF-mediatedtranscription in a cell. Wnt agonists are therefore selected from trueWnt agonists that bind and activate a Frizzled receptor family memberincluding any and all of the Wnt family proteins, an inhibitor ofintracellular β-catenin degradation, and activators of TCF/LEF. Said Wntagonist is added to the media in an amount effective to stimulate a Wntactivity in a cell by at least 10%, more preferred at least 20%, morepreferred at least 30%, more preferred at least 50%, more preferred atleast 70%, more preferred at least 90%, more preferred at least 100%,relative to a level of said Wnt activity in the absence of saidmolecule, as assessed in the same cell type. As is known to a skilledperson, a Wnt activity can be determined by measuring thetranscriptional activity of Wnt, for example by pTOPFLASH and pFOPFLASHTcf luciferase reporter constructs (Korinek et al., 1997. Science275:1784-1787).

A Wnt agonist may comprise a secreted glycoprotein includingWnt-1/Int-1; Wnt-2/Irp (Int-1-related Protein); Wnt-2b/13; Wnt-3/Int-4;Wnt-3a (R&D systems); Wnt-4; Wnt-5a; Wnt-5b; Wnt-6 (Kirikoshi H et al.2001. Biochem Biophys Res Com 283: 798-805); Wnt-7a (R&D systems);Wnt-7b; Wnt-8a/8d; Wnt-8b; Wnt-9a/14; Wnt-9b/14b/15; Wnt-10a;Wnt-10b/12; Wnt-11; and Wnt-16. An overview of human Wnt proteins isprovided in “THE WNT FAMILY OF SECRETED PROTEINS”, R&D Systems Catalog,2004.

Further Wnt agonists include the R-spondin family of secreted proteins,which is implicated in the activation and regulation of Wnt signalingpathway and which is comprised of 4 members (R-spondin 1 (NU206, Nuvelo,San Carlos, Calif.), R-spondin 2 ((R&D systems), R-spondin 3, andR-spondin-4); and Norrin (also called Norrie Disease Protein or NDP)(R&D systems), which is a secreted regulatory protein that functionslike a Wnt protein in that it binds with high affinity to the Frizzled-4receptor and induces activation of the Wnt signaling pathway (KestutisPlanutis et al. (2007) BMC Cell Biol. 8: 12).

Compounds that mimic the activity of R-spondin may be used as Wntagonists of the invention. It has recently been found that R-spondininteracts with Lgr5. Thus, Lgr5 agonists such as agonistic anti-Lgr5antibodies are examples of Wnt agonists that may be used in theinvention.

In the context of a culture medium of the invention, R-spondin is alsoreferred to herein as “R”.

A small-molecule agonist of the Wnt signaling pathway, anaminopyrimidine derivative, was recently identified and is alsoexpressly included as a Wnt agonist (Liu et al. (2005) Angew Chem Int EdEngl. 44, 1987-90).

Known GSK-inhibitors comprise small-interfering RNAs (siRNA; CellSignaling), lithium (Sigma), kenpaullone (Biomol International; Leost,M. et al. (2000) Eur. J. Biochem. 267, 5983-5994),6-Bromoindirubin-30-acetoxime (Meijer, L. et al. (2003) Chem. Biol. 10,1255-1266), SB 216763 and SB 415286 (Sigma-Aldrich), and FRAT-familymembers and FRAT-derived peptides that prevent interaction of GSK-3 withaxin. An overview is provided by Meijer et al., (2004) Trends inPharmacological Sciences 25, 471-480, which is hereby incorporated byreference. Methods and assays for determining a level of GSK-3inhibition are known to a skilled person and comprise, for example, themethods and assay as described in Liao et al 2004, Endocrinology,145(6): 2941-9).

In a preferred embodiment, said Wnt agonist is selected from one or moreof a Wnt family member, R-spondin 1-4 (for example, R-spondin 1 orR-spondin 4), Norrin, and a GSK-inhibitor. It was found by the inventorsthat the addition of at least one Wnt agonists to the basal culturemedium is important for proliferation of the liver epithelial stem cellsor isolated biliary duct or isolated liver fragments.

In a further preferred embodiment, said Wnt agonist comprises orconsists of R-spondin 1. R-spondin 1 is preferably added to the basalculture medium at a concentration of at least 200 ng/ml, more preferredat least 300 ng/ml, more preferred at least 500 ng/ml. A still morepreferred concentration of R-spondin 1 is approximately 500 ng/ml orexactly 500 ng/ml. During culturing of stem cells, said Wnt familymember may be added to the culture medium when required, for example,daily or every other day. The Wnt family member is preferably added tothe culture medium every second day. The culture medium may be refreshedwhen required, for example, daily or every other day, although ispreferably refreshed every fourth day.

In a preferred embodiment, a Wnt agonist is selected from the groupconsisting of: R-spondin, Wnt-3a and Wnt-6. More preferably, R-spondinand Wnt-3a are both used as a Wnt agonist. This combination isparticularly preferred since this combination surprisingly has asynergistic effect on organoid formation. Preferred concentrations are1-500 ng/ml, for example, 1-10 ng/ml, 10-100 ng/ml, 1-50 ng/ml, 10-200ng/ml, 200 to 500 ng/ml, 30 ng/ml for R-spondin and 100 ng/ml to 1000ng/ml, for example, 100 ng/ml or 1000 ng/ml for Wnt3a.

A Wnt agonist is preferably a Wnt ligand, such as for example Wnt3a, andmay be freshly added to a culture medium. Alternatively, a Wnt ligand isexpressed in a cell line by transfecting or infected a cell line with asuitable expression construct expressing said Wnt ligand. Said cell lineis cultured and the culture medium comprising the secreted Wnt ligand isharvested at suitable time intervals. For example, cells will produceWnt3a as soon as they reach confluency and stop growing. Culture mediumfrom cells that were not transfected or infected with said expressionconstruct is used as a control. The conditioned medium is harvested andtested, for example in an assay wherein luciferase expression incontrolled by TCF responsive elements to test for the presence of a Wntagonist such as Wnt3a (Korinek et al., 1997. Science 275:1784-1787). Themedium is diluted when used in the cultures to regenerate tissue. As isknown to the skilled person, the addition of an excess of Wnt ligandsometimes is as detrimental for the culture as is the addition of toolittle Wnt ligand. Therefore, the actual dilution of the conditionedmedium will depend on the amount of Wnt ligand that is determined in thetest.

Mitogenic Growth Factors

Yet a further component that is added to the basal culture medium is acombination of mitogenic growth factors, selected from the group ofepidermal growth factor (EGF; (preferably from Peprotech), a fibroblastgrowth factor (FGF) able to bind to FGFR2 or FGFR4, and hepatocytegrowth factor (HGF) (also preferably from Peprotech). An FGF able tobind to FGFR2 (FGF receptor) or FGFR4 is preferably FGF4, FGF7 or FGF10(preferably from Peprotech), most preferably FGF10. Preferably all 3 ofEGF, an FGF and HGF are used. In the context of a culture medium of theinvention, EGF is also referred to herein as “E”, FGF is also referredto herein as “F” and HGF is also referred to herein as “H”. EGF is apotent mitogenic factor for a variety of cultured ectodermal andmesodermal cells and has a profound effect on the differentiation ofspecific cells in vivo and in vitro and of some fibroblasts in cellculture. The EGF precursor exists as a membrane-bound molecule which isproteolytically cleaved to generate the 53-amino acid peptide hormonethat stimulates cells. EGF exerts its effects in the target cells bybinding to the plasma membrane located EGF receptor. The EGF receptor isa transmembrane protein tyrosine kinase. Binding of EGF to the receptorcauses activation of the kinase and subsequently receptorautophosphorylation. The autophosphorylation is essential for theinteraction of the receptor with its substrates. The signal transductionpathways activated by EGF include the phosphatidylinositol pathway,leading to activation of protein kinase C and to increase in theintracellular Ca2+ concentration, and to the ras pathway leading to MAPkinase activation. These pathways are involved in regulating cellularproliferation, differentiation, and survival (Herbst, 2004, Int Journalof radiation oncology, 59, 2, S21-S26).

EGF is preferably added to the basal culture medium at a concentrationof between 5 and 500 ng/ml or of at least 5 and not higher than 500ng/ml. A preferred concentration is at least 10, 20, 25, 30, 40, 45, or50 ng/ml and not higher than 500, 450, 400, 350, 300, 250, 200, 150, or100 ng/ml. A more preferred concentration is at least 50 and not higherthan 100 ng/ml. An even more preferred concentration is about 50 ng/mlor 50 ng/ml.

FGF10 is a protein that belongs to the fibroblast growth factor (FGF)family of proteins. FGF family members possess broad mitogenic and cellsurvival activities, and are involved in a variety of biologicalprocesses, including embryonic development, cell growth, morphogenesis,tissue repair, tumor growth and invasion. FGFs stimulate cells byinteracting with cell surface tyrosine kinase receptors (FGFR). Fourclosely related receptors (FGFR1-FGFR4) have been identified.FGFR1-FGFR3 genes have been shown to encode multiple isoforms, and theseisoforms can be critical in determining ligand specificity. Most FGFsbind more than one receptor (Ornitz J Biol Chem. 1998 Feb. 27; 273(9):5349-57). However, FGF10 and FGF7 are unique among FGFs in that theyinteract only with a specific isoform of FGFR2, designated FGFR2b whichis expressed exclusively by epithelial cells (Igarashi, J Biol Chem.1998 273(21):13230-5). FGF10 is a preferred FGF able to bind to FGFR2 orFGFR4.

Hepatocyte growth factor/scatter factor (HGF/SF) is a morphogenic factorthat regulates cell growth, cell motility, and morphogenesis byactivating a tyrosine kinase signaling cascade after binding to theproto-oncogenic c-Met receptor.

The same concentrations may be used for FGF10 and HGF. Preferredconcentrations for FGF10 are 20, 50, 100, 500 ng/ml, not higher than 500ng/ml. Preferred concentrations for HGF are 1, 10, 20, 50 ng/ml, nothigher than 50 ng/ml. During culturing of stem cells, said combinationof mitogenic growth factors (i.e. EGF, FGF10 and HGF) is preferablyadded to the culture medium when required, for example, daily or everyother day. It is preferable that it is added every second day. Theculture medium may be refreshed when required, for example, daily orevery other day, although is preferably refreshed every fourth day.

In some embodiments, a TGF-beta inhibitor such as A83-01 is additionallypresent in the EM1 medium. This is particularly useful when human cellsor organoids are being cultured. In some embodiments, the A83-01 ispresent at a concentration of between 400-600 nM, for example 450-550nM, 470-530 nM or approximately 500 nM. In embodiments in which aTGF-beta inhibitor is present in EM1, a Notch inhibitor is preferablynot present.

Preferred Expansion Media of the Invention

Preferred culture media and methods of using these are described in theExamples.

For example, an cell culture medium may comprise or consist of a basalmedium to which is added: EGF and R-spondin 1 supplemented with FGF10,HGF and Nicotinamide; for example, EGF (50 ng/ml) and R-spondin 1 (1ug/ml) supplemented with FGF10 (100 ng/ml), HGF (25-50 ng/ml) andNicotinamide (1-10 mM). The inventors have found that this medium ispreferable for long-term expansion of cells. Thus, this cell culturemedium is preferred for use as an EM2 of the invention. The basal mediumis preferably supplemented with B27, N2 and 200 ng/ml N-Acetylcysteine.In some embodiments, the basal medium is Advanced-DMEM/F12. However, anyother suitable basal medium may be used.

Another preferred cell culture medium, and method of using this medium,is described in the examples, and comprises or consists ofAdvanced-DMEM/F12 preferably supplemented with B27, N2, 200 ng/mlN-Acetylcysteine, 50 ng/ml EGF, 1 μg/ml R-spondin1, 10 nM gastrin, 100ng/ml FGF10, 10 mM Nicotinamide and 50 ng/ml HGF and 50% Wnt conditionedmedia and, preferably 10-100 ng/ml Noggin. Wnt conditioned mediacomprises Advanced DMEM, P/S, B27, N2 and also FCS. 293T cellstransfected with Wnt3A expression plasmid produce Wnt. The whole mediumis taken after a few days (i.e. with secreted Wnt) and used as the Wntsource.

The invention therefore provides a cell culture medium, comprising orconsisting of a basal medium for animal or human cells to which isadded:

Epidermal Growth Factor, an FGF able to bind to FGFR2 or FGFR4,preferably FGF10 and HGF as mitogenic growth factors,

gastrin, Nicotinamide, B27, N2 and N-Acetylcystein, and preferably

a BMP inhibitor more preferably Noggin and

a Wnt agonist, more preferably R-spondin 1 and/or Wnt-3a.

The invention thus encompasses a first preferred culture mediumcomprising or consisting of a basal medium for animal or human cells towhich is added:

Epidermal Growth Factor, FGF10 and HGF as mitogenic growth factors,

gastrin, Nicotinamide, B27, N2 and N-Acetylcysteine,

a BMP inhibitor more preferably Noggin and

a Wnt agonist, more preferably R-spondin 1 and Wnt-3a.

This medium may be used as an EM1 cell culture medium of the inventionto stimulate initial expansion of cells.

In some embodiments, the medium used as an EM1 cell culture mediumcomprises all the components of an EM2 culture medium of the inventionand additionally comprises Wnt-3a and Noggin.

In embodiments in which the basal medium is supplemented withN-Acetylcysteine, B27 and N2, the following are preferably added to theculture media: EGF, R-spondin1, gastrin, FGF10, Nicotinamide and HGF andWnt-conditioned media. Preferably, the basal medium is supplemented withN-Acetylcysteine, EGF, R-spondin1, gastrin, FGF10, Nicotinamide and HGFand Wnt-conditioned media in accordance with the quantities describedhereinabove.

For example, in some embodiments the basal medium may be supplementedwith 150 ng/ml to 250 ng/ml N-Acetylcysteine; preferably, the basalmedium is supplemented with, about or exactly 200 ng/mlN-Acetylcysteine. For example, in some embodiments the basal medium maybe supplemented with 40 ng/ml to 60 ng/ml EGF; preferably, the basalmedium is supplemented with about or exactly 50 ng/ml EGF. For example,in some embodiments the basal medium may be supplemented with 0.5 μg/mlto 1.5 μg/ml R-spondin1; preferably, the basal medium is supplementedwith about or exactly 1 μg/ml R-spondin1. For example, in someembodiments the basal medium may be supplemented with 5 nM to 15 nMgastrin; preferably, the basal medium is supplemented with about orexactly 10 nM gastrin. For example, in some embodiments the basal mediummay be supplemented with 25-200 ng/ml FGF10, for example 70 ng/ml to 130ng/ml FGF10; preferably, the basal medium is supplemented with about orexactly 100 ng/ml FGF10. For example, in some embodiments the basalmedium may be supplemented with 5 mM to 15 mM Nicotinamide; preferably,the basal medium is supplemented with about or exactly 10 mMNicotinamide. For example, in some embodiments the basal medium may besupplemented with 25 ng/ml to 100 ng/ml HGF, for example 35 ng/ml to 65ng/ml HGF; preferably, the basal medium is supplemented with about orexactly and 50 ng/ml HGF. For example, in some embodiments the basalmedium may be supplemented with 35% to 65% Wnt-conditioned media;preferably, the basal medium is supplemented with about or exactly 50%Wnt-conditioned media.

In some embodiments one or both of gastrin and N2 are not present in thecell culture medium.

Preferably, the basal medium is advanced-DMEM/F12.

This first culture medium (for example, EM1, EM2 or both EM1 and EM2) ispreferably used during the first two weeks of the culture method of theinvention. However, it may be used for a shorter time period, such asfor 1, 2, 3, 5, 7, or 10 days, or a longer time period, such as 3, 4, 5,10, 20 or more weeks, 5 months or more, for example, 6, 7, 8, 9, 10, 11,12 months or more.

Differentiation Medium (DM):

In another aspect, there is provided a second cell culture medium whichcomprises or consists of a basal medium for animal or human cells towhich is added: EGF, a TGF-beta inhibitor, and a Notch inhibitor. Theinventors have found that this medium is useful for differentiatingcells. The medium used for differentiating the cells may be referred toherein as DM.

Preferably, the second cell culture medium also comprises FGF and/orHGF.

In one embodiment, the second culture medium comprises or consists of abasal medium for animal or human cells to which is added:

Epidermal Growth Factor, FGF10 and HGF as mitogenic growth factors;

a Notch inhibitor; and

a TGF-beta inhibitor.

In one embodiment, the TGF-beta inhibitor is A83-01 and/or the Notchinhibitor is DAPT. In another embodiment, the DM cell culture mediumadditionally comprises Dexamethasone.

A preferred second cell culture medium, and method of using this medium,is described in the examples, and comprises or consists of a basalmedium to which is added: 50 ng/ml EGF, 100 ng/ml FGF10, 50 nM A8301 and10 uM DAPT. Advanced-DMEM/F12 may be used as the basal medium as may anyother suitable basal medium.

In some embodiments, the second cell culture medium does not compriseR-spondin or Wnt. In some embodiments, the second cell culture mediumdoes not comprise a Wnt agonist. In some embodiments, the second cellculture medium does not comprise Nicotinamide. In some embodiments, thesecond cell culture medium does not comprise a BMP inhibitor.

The inventors have discovered that R-spondin1 and Nicotinamide bothinhibit the expression of the mature hepatocyte marker CYP3A11 and yetpromote the expression of the hepatoblast marker albumin. Therefore, toincrease differentiation of the cells to more mature liver fates, theinventors removed R-spondin and Nicotinamide from the cell culture. Theinventors have also discovered that the expression of specific biliarytranscription factors is highly upregulated in expansion culturescontaining R-spondin1, indicating that the culture gene expression wasunbalanced towards a more biliary cell fate. Notch and TGF-betasignaling pathways have been implicated in biliary cell fate in vivo. Infact, deletion of Rbpj (essential to achieve active Notch signalling)results in abnormal tubulogenesis (Zong Y. Development 2009) and theaddition of TGF-beta to liver explants facilitates the biliarydifferentiation in vitro (Clotman F. Genes and Development 2005). Sinceboth Notch and TGF-beta signalling pathways were highly upregulated inthe liver cultures (FIG. 9) the inventors reasoned that inhibition ofbiliary duct cell-fate might trigger the differentiation of the cellstowards a more hepatocytic phenotype. It was found that addition of aTGF-beta inhibitor (such as A8301) and a Notch inhibitor (such as DAPT)to a differentiation medium that preferably does not contain R-spondinor Wnt, enhances the expression of mature hepatocyte markers andincreases the number of hepatocyte-like cells (for example, see example2).

Notch is a transmembrane surface receptor that can be activated throughmultiple proteolytic cleavages, one of them being cleavage by a complexof proteins with protease activity, termed gamma-secretase.Gamma-secretase is a protease that performs its cleavage activity withinthe membrane. Gamma-secretase is a multicomponent enzyme and is composedof at least four different proteins, namely, presenilins (presenilin 1or 2), nicastrin, PEN-2 and APH-I. Presenilin is the catalytic centre ofgamma-secretase. On ligand binding the Notch receptor undergoes aconformational change that allows ectodomain shedding through the actionof an ADAM protease which is a metalloprotease. This is followedimmediately by the action of the gamma-secretase complex which resultsin the release of the Notch intracellular domain (NICD). NICDtranslocates to the nucleus where it interacts with CSL(C-promoter-binding factor/recombinant signal-sequence binding proteinJκ/Supressor-of-Hairless/Lag1). The binding of NICD converts CSL from atranscriptional repressor to an activator which results in theexpression of Notch target genes. Examples of preferred Notch inhibitorsthat can be used in the context of this invention are: gamma-secretaseinhibitors, such as DAPT or dibenzazepine (DBZ) or benzodiazepine (BZ)or LY-411575, an inhibitor capable of diminishing ligand mediatedactivation of Notch (for example via a dominant negative ligand of Notchor via a dominant negative Notch or via an antibody capable of at leastin part blocking the interacting between a Notch ligand and Notch), oran inhibitor of ADAM proteases.

TGF-beta signalling is involved in many cellular functions, includingcell growth, cell fate and apoptosis. Signalling typically begins withbinding of a TGF-beta superfamily ligand to a type II receptor whichrecruits and phosphorylates a type I receptor. The type 1 receptor thenphosphorylates SMADs, which act as transcription factors in the nucleusand regulate target gene expression. Alternatively, TGF-beta signallingcan activate MAP kinase signalling pathways, for example, via p38 MAPkinase. The TGF-beta superfamily ligands comprise bone morphogenicproteins (BMPs), growth and differentiation factors (GDFs),anti-mullerian hormone (AMH), activin, nodal and TGF-betas.

A TGF-beta inhibitor is an agent that reduces the activity of theTGF-beta signalling pathway. There are many ways of disrupting theTGF-beta signaling pathway that are known in the art and that can beused in conjunction with this invention. For example, the TGF-betasignaling may be disrupted by: inhibition of TGF-beta expression by asmall-interfering RNA strategy; inhibition of furin (a TGF-betaactivating protease); inhibition of the pathway by physiologicalinhibitors, such as inhibition of BMP by Noggin, DAN or DAN-likeproteins; neutralisation of TGF-beta with a monoclonal antibody;inhibition with small-molecule inhibitors of TGF-beta receptor kinase 1(also known as activin receptor-like kinase, ALK5), ALK4, ALK6, ALK7 orother TGF-beta-related receptor kinases; inhibition of Smad 2 and Smad 3signaling by overexpression of their physiological inhibitor, Smad 7, orby using thioredoxin as an Smad anchor disabling Smad from activation(Fuchs, O. Inhibition of TGF-Signaling for the Treatment of TumorMetastasis and Fibrotic Diseases. Current Signal Transduction Therapy,Volume 6, Number 1, January 2011, pp. 29-43(15)).

Various methods for determining if a substance is a TGF-beta inhibitorare known and might be used in conjunction with the invention. Forexample, a cellular assay may be used in which cells are stablytransfected with a reporter construct comprising the human PAI-1promoter or Smad binding sites, driving a luciferase reporter gene.Inhibition of luciferase activity relative to control groups can be usedas a measure of compound activity (De Gouville et al., Br J Pharmacol.2005 May; 145(2): 166-177).

A TGF-beta inhibitor according to the present invention may be aprotein, peptide, small-molecules, small-interfering RNA, antisenseoligonucleotide, aptamer or antibody. The inhibitor may be naturallyoccurring or synthetic. Examples of preferred small-molecule TGF-betainhibitors that can be used in the context of this invention include thesmall molecule inhibitors listed in table 1:

TABLE 1 Small-molecule TGF-beta inhibitors targeting receptor kinasesIC50 Inhibitor Targets (nM) Mol Wt Name Formula A83-01 ALK5 12 421.523-(6-Methyl-2- C25H19N5S (TGF- pyridinyl)-N-phenyl-4- β R1)(4-quinolinyl)-1H- ALK4 45 pyrazole-1- ALK7 7.5 carbothioamide SB-431542ALK5 94 384.39 4-[4-(1,3-benzodioxol- C22H16N4O3 ALK45-yl)-5-(2-pyridinyl)- ALK7 1H-imidazol-2- yl]benzamide SB-505124 ALK547 335.4 2-(5-benzo[1,3]dioxol- C20H21N3O2 ALK4 129 5-yl-2-tert-butyl-3Himidazol- 4-yl)-6-methylpyridine hydrochloride hydrate SB-525334 ALK514.3 343.42 6-[2-(1,1- C21H21N5 Dimethylethyl)-5-(6-methyl-2-pyridinyl)- 1H-imidazol-4- yl]quinoxaline SD-208 ALK5 49 352.752-(5-Chloro-2- C17H10ClFN6 fluorophenyl)-4-[(4- pyridyl)amino]pteridineLY-36494 TGR-β RI 59 272.31 4-[3-(2-Pyridinyl)-1H- C17H12N4 TGF-β RII400 pyrazol-4-yl]-quinoline MLK-7K 1400 SJN-2511 ALK5 23 287.32 2-(3-(6-C17H13N5 Methylpyridine-2-yl)- 1H-pyrazol-4-yl)-1,5- naphthyridine

A method of the invention may comprise the use of one or more of any ofthe inhibitors listed in table 1. A method of the invention may comprisethe use of any combination of one inhibitor with another inhibitorlisted. For example, a method of the invention may comprise the use ofSB-525334 or SD-208 or A83-01; or a method of the invention may comprisethe use of SD-208 and A83-01; or a method of the invention may comprisethe use of SD-208 and A83-01. The skilled person will appreciate that anumber of other small-molecule inhibitors exist that are primarilydesigned to target other kinases, but at high concentrations may alsoinhibit TGF-beta receptor kinases. For example, SB-203580 is a p38 MAPkinase inhibitor that, at high concentrations (for example, approximate10 uM or more) is thought to inhibit ALK5. Any such inhibitor thatinhibits the TGF-beta signalling pathway can also be used in the contextof this invention.

A83-01 may be added to the culture medium at a concentration of between10 nM and 10 uM, or between 20 nM and 5 uM, or between 50 nM and 1 uM.For example, A83-01 may be added to the culture medium at approximately500 nM. When used in an EM, A83-01 may be added to the culture medium ata concentration of between 350-650 nM, for example, 450-550 nM, morepreferably approximately 500 nM. When used in the DM, A83-01 may beadded to the culture medium at a concentration of between 25-75 nM, forexample, 40-60 nM or approximately 50 nM.

SB-431542 may be added to the culture medium at a concentration ofbetween 80 nM and 80 uM, or between 100 nM and 40 uM, or between 500 nMand 10 uM. For example, SB-431542 may be added to the culture medium atapproximately 1 uM.

SB-505124 may be added to the culture medium at a concentration ofbetween 40 nM and 40 uM, or between 80 nM and 20 uM, or between 200 nMand 1 uM. For example, SB-505124 may be added to the culture medium atapproximately 500 nM.

SB-525334 may be added to the culture medium at a concentration ofbetween 10 nM and 10 uM, or between 20 nM and 5 uM, or between 50 nM and1 uM. For example, SB-525334 may be added to the culture medium atapproximately 100 nM.

LY 364947 may be added to the culture medium at a concentration ofbetween 40 nM and 40 uM, or between 80 nM and 20 uM, or between 200 nMand 1 uM. For example, LY 364947 may be added to the culture medium atapproximately 500 nM.

SD-208 may be added to the culture medium at a concentration of between40 nM and 40 uM, or between 80 nM and 20 uM, or between 200 nM and 1 uM.For example, SD-208 may be added to the culture medium at approximately500 nM.

SJN 2511 may be added to the culture medium at a concentration ofbetween 20 nM and 20 uM, or between 40 nM and 10 uM, or between 100 nMand 1 uM. For example, A83-01 may be added to the culture medium atapproximately 200 nM.

In a further aspect, the invention provides an alternative secondculture medium comprising or consisting of a basal medium for animal orhuman cells to which is added:

Epidermal Growth Factor, FGF10 and HGF as mitogenic growth factors;

gastrin, Nicotinamide, B27, N2 and N-Acetylcysteine.

This alternative second culture medium is useful as a differentiationmedium (DM). This alternative second culture medium is preferably usedafter the two first weeks of culture. At this stage, it seems that a BMPinhibitor and a Wnt ligand are no longer needed. Thus, in someembodiments, the alternative second culture medium does not comprise aBMP inhibitor or a Wnt ligand. In some embodiments, the alternativesecond culture medium does not comprise a BMP inhibitor or a Wntagonist.

As with the first culture medium (EM, i.e. EM1 and EM2 or EM1 or EM2),the basal medium may in some embodiments be supplemented withN-Acetylcysteine, EGF, gastrin, FGF10, Nicotinamide and HGF inaccordance with the quantities described hereinabove. For example,preferably, the basal medium is supplemented with about or exactly 200ng/ml N-Acetylcysteine. Preferably, the basal medium is supplementedwith about or exactly 50 ng/ml EGF. Preferably, the basal medium issupplemented with about or exactly 10 nM gastrin. Preferably, the basalmedium is supplemented with about or exactly 100 ng/ml FGF10.Preferably, the basal medium is supplemented with about or exactly 10 mMNicotinamide. Preferably, the basal medium is supplemented with about orexactly and 50 ng/ml HGF.

A first and a second cell culture medium used according to the inventionallows the survival and/or proliferation and/or differentiation ofepithelial stem cells or liver epithelial stem cells or isolated biliaryduct or isolated liver fragments on an extracellular matrix.

A medium allowing for survival and/or proliferation is a medium whichpreferably induces or promotes the survival and/or proliferation ofcells during at least 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100,120, 140, 160, 180, 200 days of culture. Proliferation can be assessedusing techniques known in the art such as BrdU staining, Edu staining,Ki67 staining and the use of growth curves assay can be done. Forexample, we have shown that from 10 biliary ducts, it is possible after6 days to dilute to 6 wells with 10 organoids per well (60 neworganoids). In each passage of 6 days we can generate around 360organoids. By performing 1 passage/week over 32 weeks we are able togenerate over 11,000 (11520) new organoid structures in only 7 months.This is important for the industry, since the availability of cells andorganoids for transplantation poses a significant problem. For a mousetransplant, for example, a minimum of 10⁵ cells are required. Possibly10⁶, or 10×10⁶ might be required for a human transplant, in order for agraft to be successful.

Put another way, media used according to the invention is capable ofexpanding a population of stem cells to form liver organoids for atleast 3, at least 4, at least 5, at least 6, at least 7, at least 8, atleast 9, at least 10, at least 20, at least 30, at least 40, at least50, at least 60, at least 70, at least 80, at least 90 or at least 100,passages under appropriate conditions.

The second medium as identified above preferably induces or promotes aspecific differentiation of cells during at least five days of culture.Differentiation may be measured by detecting the presence of a specificmarker associated with the liver lineage as defined herein.Differentiation may be measured by detecting the presence of a specificmarker associated with the liver lineage as defined herein. Depending onthe identity of the marker, the expression of said marker may beassessed by RTPCR or immuno-histochemistry after at least 5, 7, 8, 9,10, 11, 12, 13, 14, 15, 16 days of culture in a first medium or in afirst subsequently in a second medium as defined herein.

A differentiation medium is a medium which preferably induces orpromotes a specific differentiation of cells during at least five daysof culture. Differentiation may be measured by detecting the presence ofa specific marker associated with the liver lineage as defined herein.

Within the context of the invention, the term cell culture medium issynonymous with medium, culture medium or cell medium or basal medium orbasal cell medium or basal cell culture medium or expansion medium ordifferentiation medium.

According to a still further aspect of the invention, there is provideda hermetically-sealed vessel containing a culture medium of theinvention. In some embodiments, the culture medium is an expansionmedium. In some embodiments, the culture medium is a differentiationmedium. Hermetically-sealed vessels may be preferred for transport orstorage of the culture media, to prevent contamination. The vessel maybe any suitable vessel, such as a flask, a plate, a bottle, a jar, avial or a bag.

Methods for Obtaining and/or Culturing Stem Cells

The method for obtaining and/or culturing a liver fragment or a liverorganoid, comprises culturing epithelial stem cells, and/or isolatedtissue fragments comprising said epithelial stem cells in contact withan extracellular matrix in the presence of one or more cell culturemedia according to the invention.

In some embodiments, a method for obtaining and/or culturing a liverfragment or a liver organoid comprises culturing epithelial stem cells,and/or isolated tissue fragments comprising said epithelial stem cellsin contact with an extracellular matrix in the presence of EM1 mediumand then EM2 medium and then DM medium.

In some embodiments, a method for obtaining and/or culturing a liverfragment or a liver organoid comprises culturing epithelial stem cells,and/or isolated tissue fragments comprising said epithelial stem cellsin contact with an extracellular matrix in the presence of EM2 mediumand then DM medium without the use of EM1 medium.

In some embodiments, a method for obtaining and/or culturing a liverfragment or a liver organoid comprises culturing epithelial stem cells,and/or isolated tissue fragments comprising said epithelial stem cellsin contact with an extracellular matrix in the presence of EM1 mediumand then DM medium without the use of EM2 medium.

The method for obtaining and/or culturing a liver fragment or a liverorganoid, may comprise:

i) culturing epithelial stem cells, and/or isolated tissue fragmentscomprising said epithelial stem cells in contact with an extracellularmatrix in the presence of a medium, the medium comprising a basal mediumfor animal or human cells to which is added: Epidermal Growth Factor, anFGF able to bind to FGFR2 or FGFR4, preferably FGF10 and HGF asmitogenic growth factors, Nicotinamide, and preferably, a Wnt agonist,preferably any one of R-spondin 1-4; and subsequently

ii) culturing the stem cells, and/or isolated tissue fragmentscomprising said epithelial stem cells in contact with an extracellularmatrix in the presence of a second cell culture medium as defined here(a DM medium).

In some embodiments, prior to the step i), the method comprisesculturing the epithelial stem cells, and/or isolated tissue fragmentscomprising said epithelial stem cells in contact with an extracellularmatrix in the presence of a medium comprising or consisting of a basalmedium for animal or human cells to which is added EGF, a BMP inhibitor,R-spondin and Wnt. Preferably, the BMP inhibitor is Noggin and the EM1medium is termed “ENRW” (EGF, Noggin, R-spondin and Wnt).

In one embodiment there is provided a method for obtaining and/orculturing a liver fragment or a liver organoid, wherein said methodcomprises:

culturing epithelial stem cells, and/or isolated tissue fragmentscomprising said epithelial stem cells in contact with an extracellularmatrix in an EM2 culture medium as described herein, for example, amedium comprising a Wnt agonist, such as R-spondin, Epidermal GrowthFactor, an FGF able to bind to FGFR2 or FGFR4, preferably FGF10, andnicotinamide. Preferably, the medium also comprises HGF.

In a further embodiment there is provided a method for obtaining and/orculturing a liver fragment or a liver organoid, wherein said methodcomprises:

culturing epithelial stem cells, and/or isolated tissue fragmentscomprising said epithelial stem cells in contact with an extracellularmatrix in a medium comprising a BMP inhibitor, a Wnt agonist, EpidermalGrowth Factor, a FGF able to bind to FGFR2 or FGFR4, preferably FGF10and HGF as mitogenic growth factors, gastrin, Nicotinamide, B27, N2 andN-Acetylcysteine.

In another preferred method, after at least two weeks of culture in anEM1 medium as defined above, the culture is continued in a medium thatdoes not comprise a BMP inhibitor and a Wnt agonist. In someembodiments, this culture is continued in an EM2 of the invention. Inalternative embodiments, the culture is continued in a second medium ofthe invention (DM), which additionally comprises a TGF-beta inhibitor,such as A83-01, and a Notch inhibitor, such as DAPT. In someembodiments, this culture is continued in an EM2 and then a DM medium ofthe invention.

Therefore in a preferred method for obtaining and/or culturing a liverorganoid, epithelial stem cells, and/or isolated liver tissue fragmentscomprising said epithelial stem cells are cultured in a first step inthe first medium (EM), then subsequently in a discrete second step inthe second medium (DM). The first culture step may have a duration of atleast two weeks and may be longer, for example, 3 weeks or longer, 4weeks or longer or 1, 2, 3, 4, 5, 6, 7, 8 months or longer. The secondstep preferably has a duration of 6 weeks or less, more preferably 1month or less, for example 3 weeks or less, 2 weeks or less, 1 week orless. It has been found that many cells start dying after 2 weeks in DMand so preferably, the second step is 2 weeks or less or at most 1month. Thus, it is only in less preferred embodiments, that the secondstep may have a duration of 8, 9, 10, 11, 12, 13, 14, 15, 16 days or 3weeks or 1, 2, 3, 4, 5, 6 months or longer. Each step is preferablycarried out using an extracellular matrix as defined herein, and usingthe culture media described in detail herein.

Alternatively in another embodiment, said method is carried out in thefirst medium, without switch to the second medium. This method may havea duration of 8, 9, 10, 11, 12, 13, 14, 15, 16 days or 3 weeks or 1, 2,3, 4, 5, 6, 7, 8 months or longer.

In some embodiments, the step of culturing in the first medium of theinvention comprises both culturing in EM1 medium and then culturing inEM2 medium. Thus, the periods described above may refer to the totalperiod in which the cells or fragments are cultured in the EM1 andsubsequently in the EM2 media. In some embodiments, the step ofculturing in the EM1 culture medium may have a duration of less than 10days, for example, less than 9, less than 8, less than 7, less than 6,less than 5, less than 4, less than 3, less than 2 or less than 1 day.In some embodiments, the step of culturing in the EM1 culture medium mayhave a duration of between 1 and 10 days, for example, 1-7 days, 2-6days, 3-5 days, for example 4 or 5 days. In some embodiments, the stepof culturing in the EM2 medium may have a duration of 10 days or longer,for example, 11, 12, 13, 14, 15, 16 days or longer, or 1-3 weeks or 2-4weeks or 3-6 weeks or 1, 2, 3, 4, 5, 6 months or longer.

Preferred concentrations of each compound present in each medium havealready been defined herein in the description or in the examples.

The methods described herein for obtaining and/or culturing a liverfragment or a liver organoid may also be used to obtain and/or culture apopulation of cells expressing Lgr5 and such methods are encompassed bythe invention. A population of cells expressing Lgr5 obtained by amethod of the invention is thus also provided.

As will be apparent to the skilled reader, the preferred culture methodsof the invention are advantageous because feeder cells are not required.Feeder cell layers are often used to support the culture of stem cells,and to inhibit their differentiation. A feeder cell layer is generally amonolayer of cells that is co-cultured with, and which provides asurface suitable for growth of, the cells of interest. The feeder celllayer provides an environment in which the cells of interest can grow.Feeder cells are often mitotically inactivated (e.g. by irradiation ortreatment with mitomycin C) to prevent their proliferation. The use offeeder cells is undesirable, because it complicates passaging of thecells (the cells must be separated from the feeder cells at eachpassage, and new feeder cells are required at each passage). The use offeeder cells can also lead to contamination of the desired cells withthe feeder cells. This is clearly problematic for any medicalapplications, and even in a research context, complicates analysis ofthe results of any experiments performed on the cells. As notedelsewhere herein, the culture media of the invention are particularlyadvantageous because they can be used to culture cells without feedercell contact, i.e. the methods of the invention do not require a layerof feeder cells to support the cells whose growth is being sponsored.

Accordingly, the compositions of the invention may be feeder cell-freecompositions. A composition is conventionally considered to be feedercell-free if the liver cells in the composition have been cultured forat least one passage in the absence of a feeder cell layer. A feedercell-free composition of the invention will normally contain less thanabout 5%, less than about 4%, less than about 3%, less than about 2%,less than about 1% feeder cells (expressed as a % of the total number ofcells in the composition) or preferably no feeder cells at all.

In one preferred embodiment, for example, if a liver organoid is to beused for regenerative medicine, the method may start from epithelialcells or from an isolated liver fragment. The cells or liver fragmentmay be autologous or allogeneic. “Autologous” cells are cells whichoriginated from the same organism into which they are beingre-introduced for cellular therapy, for example in order to permittissue regeneration. An autologous cell does not, in principle, requirematching to the patient in order to overcome the problems of immunerejection, and/or reduces the need for immune suppression interventionsupon transplant. “Allogeneic” cells are cells which originated from anindividual which is different from the individual into which the cellsare being introduced for cellular therapy, for example in order topermit tissue regeneration, although of the same species. Some degree ofpatient matching may still be required to prevent the problems ofrejection. Techniques for minimising tissue rejection will be known tothose of skill in the art.

A preferred method of the invention encompasses a method for culturingan isolated liver fragment comprising said epithelial stem cells.

Another preferred method encompasses a method for obtaining a liverorganoid from epithelial stem cells and/or isolated liver fragmentscomprising said epithelial stem cells.

Another preferred method encompasses a method for obtaining andculturing a liver organoid from epithelial stem cells and/or isolatedliver fragments comprising said epithelial stem cells.

In a preferred method as earlier identified herein, the BMP inhibitor isselected from Noggin, DAN, and DAN-like proteins including Cerberus andGremlin, preferably comprises or is Noggin, and/or said Wnt agonist isselected from one or more of Wnt, preferably Wnt-3a, R-spondin 1-4,Norrin, and a GSK-inhibitor, more preferably comprises or is Wnt-3aand/or R-spondin.

In another preferred method, a liver organoid originates from one singlecell, preferably a cell expressing Lgr5, more preferably wherein thesingle cell comprises a nucleic acid construct comprising a nucleic acidmolecule of interest. The cell may also express liver-specific markerssuch as Hnf1α, and Hnf4.

The isolation of certain cell types expressing Lgr5 has already beendescribed previously (see, for example, WO2009/022907 andWO2010/016766). However, liver specific stem cells expressing Lgr5 ofthe type disclosed herein have not previously been described.Accordingly, the invention provides a population of adult stem cellscharacterised by natural expression of at least Lgr5 and one or more ofthe following markers Hnf1α, Hnf4a, Sox9, KRT7 and KRT19 at asignificant level. This cell population also expresses markers ofprogenitor populations common to the small intestine and stomach, suchas Cd44 and Sox9 (Barker & Huch et al Cell stem cell 2010). These arehighly expressed in the stem cells according to the invention, but arenot expressed in adult liver, reinforcing the self-renewal capacity ofthe liver cultures described herein. Cells according to this aspect ofthe invention may also up-regulate Wnt target genes, including forexample, MMP7, Sp5 and Tnfrs19. This provides strong evidence of therequirement for an active and robust canonical Wnt signalling activityto maintain the self renewing capacity of these cultures.

By “natural expression” is meant that the cells have not beenmanipulated recombinantly in any way, i.e., the cells have not beenartificially induced to express these markers or to modulate thesemarkers' expression by introduction of exogenous genetic material, suchas introduction of heterologous (non-natural) or stronger promoters orother regulatory sequences operably linked to either the endogenousgenes or exogenously-introduced forms of the genes. Natural expressionis from genomic DNA within the cells, including introns between the exoncoding sequences where these exist. Natural expression is not from cDNA.Natural expression can if necessary be proven by any one of variousmethods, such as sequencing out from within the reading frame of thegene to check that no extraneous heterogenous sequence is present.“Adult” means post-embryonic. With respect to the stem cells of thepresent invention, the term “adult stem cell” means that the stem cellis isolated from a tissue or organ of an animal at a stage of growthlater than the embryonic stage.

This stem cell population can also be characterised by a lack of naturalexpression of certain markers at any significant level, many of whichare associated with cellular differentiation. Specifically, the cells ofthe isolated adult stem cell population do not naturally express one ormore of Cd11b, CD13, CD14, AFP, Pdx1, any CYP member (e.g. CYP3A11, CYP11A1) at a significant level. As defined herein, these markers are saidbe to be negative markers.

The term “expressed” is used to describe the presence of a marker withina cell. In order to be considered as being expressed, a marker must bepresent at a detectable level. By “detectable level” is meant that themarker can be detected using one of the standard laboratorymethodologies such as PCR, blotting or FACS analysis. A gene isconsidered to be expressed by a cell of the population of the inventionif expression can be reasonably detected after 30 PCR cycles, whichcorresponds to an expression level in the cell of at least about 100copies per cell. The terms “express” and “expression” have correspondingmeanings. At an expression level below this threshold, a marker isconsidered not to be expressed. The comparison between the expressionlevel of a marker in a cell of the invention, and the expression levelof the same marker in another cell, such as for example an embryonicstem cell, may preferably be conducted by comparing the two cell typesthat have been isolated from the same species. Preferably this speciesis a mammal, and more preferably this species is human. Such comparisonmay conveniently be conducted using a reverse transcriptase polymerasechain reaction (RT-PCR) experiment.

Any one of a number of physical methods of separation known in the artmay be used to select the cells of this aspect of the invention anddistinguish these from other cell types. Such physical methods mayinvolve FACS and various immuno-affinity methods based upon makersspecifically expressed by the cells of the invention. As describedabove, Lgr5, Hnf1α and Hnf4 are 3 of the cell markers expressed at highlevels in the cells of the invention. Therefore, by way of illustrationonly, the cells of the invention may be isolated by a number of physicalmethods of separation, which rely on the presence of these markers.

In one embodiment, the cells of the invention may be isolated by FACSutilizing an antibody, for example, against one of these markers. Aswill be apparent to one skilled in the art, this may be achieved througha fluorescent labeled antibody, or through a fluorescent labeledsecondary antibody with binding specificity for the primary antibody.Examples of suitable fluorescent labels includes, but is not limited to,FITC, Alexa Fluor® 488, GFP, CFSE, CFDA-SE, DyLight 488, PE, PerCP,PE-Alexa Fluor® 700, PE-Cy5 (TRI-COLOR®), PE-Cy5.5, PI, PE-Alexa Fluor®750, and PE-Cy7. This list is provided by way of example only, and isnot intended to be limiting.

It will be apparent to a person skilled in the art that FACS analysisusing an anti-Lgr5 antibody will provide a purified cell population.However, in some embodiments, it may be preferable to purify the cellpopulation further by performing a further round of FACS analysis usingone or more of the other identifiable markers, preferably Hnf1α andHnf4, but others may also be used.

In another embodiment, the cells of the invention may be isolated byimmuno-affinity purification, which is a separation method well known inthe art. By way of illustration only, the cells of the invention may beisolated by immuno-affinity purification directed towards c-kit. As willbe apparent to one skilled in the art, this method relies upon theimmobilisation of antibodies on a purification column. The cell sampleis then loaded onto the column, allowing the appropriate cells to bebound by the antibodies, and therefore bound to the column. Following awashing step, the cells are eluted from the column using a competitorwhich binds preferentially to the immobilised anti-c-kit antibody, andpermits the cells to be released from the column.

It will be apparent to a person skilled in the art that immuno-affinitypurification using an immobilised antibody will provide a purified cellpopulation. However, in some embodiments, it may be preferable to purifythe cell population further by performing a further round ofimmuno-affinity purification using one or more of the other identifiablemarkers, for example Hnf4, and use an aliquot of the isolated clones toascertain the expression of other relevant intracellular markers.

It will be apparent to a person skilled in the art that the sequentialpurification steps are not necessarily required to involve the samephysical method of separation. Therefore, it will be clear that, forexample, the cells may be purified through a FACS step using ananti-Lgr5 antibody, followed by an immuno-affinity purification stepusing a SSEA-1 affinity column. In certain embodiments, the cells may becultured after isolation for at least about 15, at least about 20 days,at least about 25 days, or at least about 30 days. In certain aspects,the cells are expanded in culture longer to improve the homogeneity ofthe cell phenotype in the cell population.

Other features of this method are defined in the part of the descriptiondedicated to definitions. Single-cell suspensions or small clusters ofcells (2-50 cells/cluster) will normally be seeded, rather than largeclusters of cells, as in known in the art. As they divide, such cellswill be seeded onto a support at a density that promotes cellproliferation. Typically, when single cells are isolated the platingdensity of at least 1-500 cells/well is used, the surface of the wellbeing 0.32 cm². When clusters are seeded the plating density ispreferably 250-2500 cells/cm². For replating, a density of between about2500 cells/cm² and about 5,000 cells/cm² may be used. During replating,single-cell suspensions or small cluster of cells will normally beseeded, rather than large clusters of cells, as in known in the art.

Organoids of the Invention

The cells described above grow into bodies which are herein termed“organoids”. Accordingly, a liver organoid obtainable by a method of theinvention is a further aspect of the invention. To the best of ourknowledge, this is the first time that a liver organoid has beenobtained that is functional and alive after such an extended period oftime (i.e. at least 7 months of culture; see examples included herein).Functionality is preferably characterized by the presence of a livermarker as defined herein and/or by the structure of said organoid asdefined herein. Since the final amount of liver organoids obtainedcorrelates with the duration of culture, the skilled person willunderstand that the invention is a pioneer invention and potentiallyopens new possibilities in for example regenerative medicine.

For example, an organoid according to the present invention may comprisea population of cells of at least 1×10³ cells, at least 1×10⁴ cells, atleast 1×10⁵ cells, at least 1×10⁶ cells, at least 1×10⁷ cells or more.In some embodiments, each organoid comprises between approximately 1×10³cells and 5×10³ cells; generally, 10-20 organoids may be grown togetherin one well of a 24 well plate.

Cells and organoids according to the present invention may be non-humananimal or human. The inventors have shown, for the first time, that itis possible to grow and maintain both animal and human liver organoidsin vitro, using the culture media and methods of the invention.

Illustrative examples of organoids generated according to the inventionare given in the accompanying figures. It can be seen that organoidsaccording to the invention may possess a cystic structure, with on theoutside, a layer of cells with at least one bud and a central lumen. Theorganoids in the outside of the MATRIGEL™ tend to be larger than theorganoids in the center of the MATRIGEL™, perhaps because they havebetter access to the necessary growth factors. Structurally, organoidsaccording to the invention are often elongated in shape. They mayinclude one or more budding structure—a single cell epithelial layerwhich has a structure not unlike a bile duct. Under confocal microscopy,the structures may stain positive for keratin. They may include cellswith polarised nuclei and small cytoplasm. The organoids may have asection which is formed of multiple layers; such cells often tend tohave their nuclei more central to the cells, i.e. not polarized. Thecells in the multilayer section may organise themselves to include agap, or lumen between the cells.

A liver organoid preferably comprises a hepatocyte and a cholangiocytecell, more preferably wherein at least one of the following markerscould be detected: at least one hepatocyte marker such as albumin,transthyretrin, B-1 integrin and Glutamine synthetase and/or at leastone of CYP3A11, FAH, tbx3, TAT and Gck and/or at least one cholangiocytemaker such as Keratin 7 and 19. The skilled person knows how to detecteach of these markers (i.e. RT-PCR and/or immunohistochemistry).Preferably the expression of each of these markers is assessed ascarried out in the experimental part. Each of these markers is usuallyexpressed after at least two weeks, three weeks or one month of cultureusing a method of the invention. Microarray analysis of the organoids inboth culture conditions showed that liver organoids resemble adult livertissue.

In some embodiments, approximately 35% of the cells in a liver organoidexpress a hepatocyte surface marker, for example, 25-45%, 30-40%,33-37%, 35% or less, or 15-35% of cells.

Preferably, cells and organoids generated according to the inventionalso possess hepatocyte functions, such as expressing or stainingpositive for the mature hepatic markers albumin, B-1 integrin, CK-8,CK-18, transthyretin (TTR), glucose 6P, Met, Glutamine synthase (Glu1),transferrin, Fahd1, Fahd2a, K7, K19 and cytochrome P450 isoforms 3A13(CYP3A13), 51 (CYP51) 2D10 (CYP2D10), 2j6 (CYP2j6), 39A1 (CYP39A1), 4A10(CYP4A10), 4F13 (CYP4F13) 4F16 (CYP4F16), CYP4B1 and 20A1(CYP20A1).Also, embryonic liver gene AFP is in some embodiments not detected inneither of both culture conditions, as in adult liver. In someembodiments, the expression of alpha fetal protein is just above thebackground gene expression.

Also, the well known liver transcription factors as HNF1a, HNF1b andHNF4a are highly expressed in both conditions.

Since liver and pancreas are closely related organs, we investigatedwhether our liver cultures also expressed pancreas-specific genes. Thepancreas is functionally divided into endocrine and exocrine pancreas.The endocrine pancreas is mainly characterized for expressing insulin,glucagon and somatostatin. The expression of these hormones is tightlyregulated by a set of endocrine pancreas-specific transcription factors,the most important being Pdx1 and NeuroD. The exocrine pancreas isformed by acinar and ductal compartments responsible of producing thedigestive enzymes amylase, pancreatic lipase and chymotrypsin, amongothers. The expression of these genes is also regulated by specificexocrine pancreatic genes as Ptf1.

The pancreas specific genes Ptf1a, pancreatic amylase (Amy2a4),pancreatic lipase (Pnlip), insulin (ins1 and ins2), glucagon (Gcg),chymotrypsin (cela1), Pdx1 and NeuroD were absent in the liver cultureshere described.

In some embodiments, one or more or all of the following genes areexpressed in the liver organoids at a similar level to the correspondinggene in adult liver hepatocytes: Aqp1, Bmp2, Apo3, Apo17a, Sord, C3,Ppara, Pparg, tbx3, lgf1, 1117rb, 111b, Tgfbi, Apoa1, Apoa4, Apob,Cyp26b1, Cyp27a1, Cyp2b13, Cyp2b9, Cyp2c37, Cyp2f2, Cyp2g1, Cyp2j13,Cyp3a11, Cyp4a10 and Cypf14. For example, see FIG. 16A.

In some embodiments, one or more of the following genes is expressed inthe liver organoids at a similarly shut down level compared to thecorresponding gene in adult liver hepatocytes: Cc12, Osmr, Icam1 andCxcl2.

In some embodiments, one or both of the following genes isdifferentially expressed in both a liver organoid and newborn liver:mKi67 and cdkn3.

In some embodiments, one, two or all of the following genes areexpressed at a similar level in a liver organoid and a newborn liver:cyp2j6, olfm4 and Lefty 1. For example, see FIG. 16B.

In some embodiments, a liver organoid of the invention has a ductalphenotype when cultured in expansion medium of the invention (e.g. EM1or EM2).

In some embodiments, a liver organoid of the invention expresses adultliver markers when cultured in a differentiation medium of theinvention.

In one embodiment, a liver organoid of the invention has a geneexpression profile as shown in FIG. 16C.

In a particularly preferred embodiment, a mouse liver cell population ororganoid of the invention has the gene expression profile as shown inFIG. 18. For example, in one preferred embodiment, a mouse liver cellpopulation or organoid of the invention:

a) expresses at least one (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11),preferably all of the following stem cell markers: 1gr5, 1gr4, epcam,Cd44, Tnfrsf19, Sox9, Spy, Cd24a, Prom1, Cdca7 and Elf3; and/or

b) does not express the following stem cell marker: 1gr6; and/or

c) expresses at least one (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19), preferably all of the following hepatocyteor cholangiocyte markers when grown in expansion medium of theinvention: Hnf1a, Hnf1b, Hnf4a, Hhex, Onecut1, Onecut2, Prox1, Cdh1,Foxa2, Gata6, Foxm1, Cebpa, Cebpb, Cebpd, Cebpg, Glu1, Krt7, Krt19 andMet; and/or

d) does not express at least one (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17) of the following genes when grown inexpansion medium of the invention: afp, Ins1, Ins2, Gcg, Ptf1a, Cela1,Cela2a, Cela3b, Neurod1, Neurod2, Neurog1, Neurog2, Neurog3, Amy2a4,Igf1r, Igf2 and Cd34; and/or

e) expresses at least one (e.g. 1, 2 or 3) of the followingreprogramming genes: Klf4, Myc and Pou5f1 and/or

f) does not express the following reprogramming gene: Sox2.

wherein the expression of the genes is preferably detected by measuringexpression at the mRNA level, for example, using a microarray.

More preferably a mouse liver cell population or organoid of theinvention has all of features a) to f) above.

In some embodiments, the gene expression profile described above for amouse cell population or organoid of the invention is for a mouse cellpopulation or organoid cultured in expansion medium of the invention.

In some embodiments, there is provided a human liver cell population ororganoid of the invention that has the gene expression signature shownin FIG. 19. For example, a human liver cell population or organoidcultured in EM1 of the invention preferably expresses the genesindicated in FIG. 19 as being expressed in EM1 cell culture medium. Forexample, a human liver cell population or organoid cultured in EM2 ofthe invention preferably expresses the genes indicated in FIG. 19 asbeing expressed in EM2 cell culture medium. For example, a human livercell population or organoid cultured in DM of the invention preferablyexpresses the genes indicated in FIG. 19 as being expressed in DM cellculture medium.

For example, in one preferred embodiment, a human liver cell populationor organoid of the invention:

a) expresses at least one (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9), preferablyall of the following stem cell signature genes: LGR4, TACSTD1/Epcam,CD44, SOX9, SP5, CD24, PROM1, CDCA7 and ELF3; and/or

b) expresses at least one (e.g. 1, 2, 3, 4), preferably all of thefollowing reprogramming genes: KLF4, MYC, POU5F1 and SOX2; and/or

c) expresses at least one (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19), preferably all of the followinghepatocyte/cholangiocyte specific genes: HNF1A, HNF1B, HNF4A, HHEX,ONECUT1, ONECUT2, PROX1, CDH1, FOXA2, GATA6, FOXM1, CEBPA, CEBPB, CEBPD,CEBPG, GLUL, KRT7, KRT19 and MET; and/or

d) does not express at least one (e.g. 1, 2, 3, 4, 5, 6), preferably allof the following hepatocyte/cholangiocyte specific genes: NEUROG2, IGF1Rand CD34, AFP, GCG and PTF1A, for example, it does not express NEUROG2,IGF1R and CD34; and/or

e) expresses at least one (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18), preferably all of the following hepatocytespecific genes: TTR, ALB, FAH, TAT, CYP3A7, APOA1, HMGCS1, PPARG,CYP2B6, CYP2C18, CYP2C9, CYP2J2, CYP3A4, CYP3A5, CYP3A7, CYP4F8, CYP4V2and SCARB1;

wherein the expression of the genes is preferably detected by measuringexpression at the mRNA level, for example, using a microarray.

More preferably a human liver cell population or organoid of theinvention has all of features a) to e) above.

In some embodiments, the genes in a human liver cell population ororganoid of the invention are upregulated or downregulated relative toexpression of a reference RNA as shown in FIG. 19. Preferably, thereference RNA is Universal Human Reference RNA (Stratagene, Catalog#740000). In some embodiments, a gene is upregulated or downregulatedrelative to the reference RNA if it is also shown in FIG. 19 as beingupregulated or downregulated relative to the reference RNA but theextent of upregulation or downregulation need not be the same. In otherembodiments, the extent of upregulation or downregulation is +/−35%,+/−30%, +/−25%, +/−20%, +/−20%, +/−15%, +/−10%, +/−5%, +/−3 orapproximately the same as shown in FIG. 19. In other embodiments, theabsolute level of expression of the genes in a human organoid of theinvention is +/−35%, +/−30%, +/−25%, +/−20%, +/−15%, +/−10%, +/−5%,+/−3% or approximately the same as shown in FIG. 19.

The human liver cell population or organoids of the invention alsopreferably express Lgr5 and/or Tnfrsf19, preferably both. In someembodiments, the human liver cell population or organoids, when culturedin expansion medium of the invention express Lgr5 and/or Tnfrsf19,preferably both. Preferably, expression of Lgr5 and/or Tnfrsfr19 isdetected by RT PCR. In some embodiments, Lgr5 and/or Tnfrsf19 arepresent at much lower levels of expression in organoids or cells whencultured in the differentiation medium compared to their level ofexpression organoids or cells when cultured in the expansion medium (forexample at least 2-fold, at least 3-fold, at least 4-fold, at least5-fold, at least 10-fold, at least 15-fold lower).

Cells and organoids according to the present invention may preferably becapable of secreting albumin, for example, at a rate of betweenapproximately 1 μg per hour per 10⁶ cells and 10 μg per hour per 10⁶cells, preferably between 2 μg and 6 μg per hour per 10⁶ cells.

Furthermore, such cells and organoids may secrete urea. For example, ina 35 mm dish of cells, the activity of urea synthesis may be between 1μg and 50 μg in 48 hours, preferably between 5 μg and 30 μg.

Cells and organoids according to the invention may show visible glycogenstores, for example, when stained. The capacity for cells and organoidsaccording to the invention to synthesize glycogen actively can be testedby switching the culture media from low-glucose differentiation media tohigh-glucose DMEM supplemented with 10% FBS and 0.2μM dexamethasone fortwo days.

Cells and organoids according to the invention may possess induciblecytochrome P450 activity (e.g. CYP1A). Such activity may be tested, forexample, using an ethoxyresorufin-O-deethylase (EROD) assay (Cancer Res,2001, 61: 8164-8170). For example, cells or organoids may be exposed toa P450 substrate such as 3-methylcholanthrene and the levels of ERODactivity compared to control cells.

Morphologically, the cells appear hepatocyte-like.

A preferred liver organoid comprises or consists of a cystic structurewith on the outside a layer of cells with buds and a central lumen asdepicted in FIG. 2. This liver organoid may have one or more (e.g. 2, 3,or all 4) of the following characteristics: (a) having a cell density of>5×10⁵ cells/cm³, preferably >10×10⁵ cells/cm³; (b) having a thicknessequivalent to 2-30 layers of cells, preferably a thickness equivalent to2-15 layers of cells; (c) the cells mutually contact in threedimensions, (d) demonstrate a function inherent to healthy liver tissue,(e) have an elongated shape, with 2 defined domains, i.e. a singlelayered epithelial domain where highly polarized cells are detected andkeratin markers are expressed (this domain resembles the bile ductdomain) and the other domain constitutes the main body of the organoidand is formed by a multilayered epithelia with non-polarized cellswherein albumin expression may be detected. It is clear to the skilledperson that such a liver organoid is preferably not a liver fragmentand/or does not comprise a blood vessel, and/or does not comprise aliver lobule or a bile duct.

Within the context of the invention, a liver fragment is a part of anadult liver, preferably a human adult liver. Preferably a liver organoidas identified herein is therefore not a liver fragment. A liver organoidis preferably obtained using a cell from an adult liver, preferably anepithelial stem cell from an adult liver, more preferably an epithelialstem cell from an adult liver expressing Lgr5.

In some embodiments, a liver organoid comprises cells that express Lgr5.For example, in some embodiments, at least 2%, more preferably at least5%, at least 10%, at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95% of the cells in the liver organoid express Lgr5. Similarly, theinvention provides a cell or a population of cells which express Lgr5,wherein said cells are obtained from a liver organoid of the invention.The progeny of such cells is also encompassed by the invention.

In an embodiment, a liver organoid is a liver organoid which is stillbeing cultured using a method of the invention and is therefore incontact with an extracellular matrix. Preferably, a liver organoid isembedded in a non-mesenchymal extracellular matrix. Within the contextof the invention, “in contact” means a physical or mechanical orchemical contact, which means that for separating said liver organoidfrom said extracellular matrix a force needs to be used.

In a preferred embodiment, a liver organoid could be cultured during atleast 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10months or longer.

In another preferred embodiment, a liver organoid originates from asingle cell, preferably expressing Lgr5, more preferably wherein thesingle cell comprises a nucleic acid construct comprising a nucleic acidmolecule of interest.

The invention further provides the use of a culture medium according tothe invention for culturing epithelial stem cells or isolated organoidstructures that comprise these stem cells on an extracellular matrix,whereby said stem cells preferably do not comprise human embryonic stemcells. Preferred are human adult stem cells. Furthermore, single sortedepithelial stem cells from the liver are also able to initiate these 3dimensional organoids in a culture medium according to the invention.The invention further provides the use of a culture medium according tothe invention for culturing liver fragments comprising stem cells.

It is preferred that said stem cells are liver stem cells, or epithelialstem cells. A culture medium according to the invention allowed theestablishment of long-term culture conditions under which a liverorganoid is formed in which all differentiated cell types are present.Using a culture method according to the invention allowed cultureperiods of at least seven months, at least eight months, at least ninemonths, at least ten months.

The invention further provides a liver organoid, preferably comprisingat least 50% viable cells, more preferred at least 60% viable cells,more preferred at least 70% viable cells, more preferred at least 80%viable cells, more preferred at least 90% viable cells. Viability ofcells may be assessed using Hoechst staining or Propidium Iodidestaining in FACS.

The viable cells preferably possess hepatic functions, orcharacteristics of hepatocytes, as described above.

Uses of Cells and Organoids of the Invention

In a further aspect, the invention provides the use of a liver cell ororganoid according to the invention as described above in a drugdiscovery screen, toxicity assay or in regenerative medicine. Theinvention furthermore provides the use of the progeny of liver organoidsof the invention, in toxicity assays. Such toxicity assays may be invitro assays using a cell derived from a liver organoid or a liverorganoid or part thereof. Such progeny and liver organoids are easy toculture and more closely resemble primary epithelial cells than, forexample, epithelial cell lines such as Caco-2 (ATCC HTB-37), I-407 (ATCCCCL6), and XBF (ATCC CRL 8808) which are currently used in toxicityassays. It is anticipated that toxicity results obtained with liverorganoids more closely resemble results obtained in patients. Acell-based toxicity test is used for determining organ specificcytotoxicity. Compounds that are tested in said test comprise cancerchemopreventive agents, environmental chemicals, food supplements, andpotential toxicants. The cells are exposed to multiple concentrations ofa test agent for certain period of time. The concentration ranges fortest agents in the assay are determined in a preliminary assay using anexposure of five days and log dilutions from the highest solubleconcentration. At the end of the exposure period, the cultures areevaluated for inhibition of growth. Data are analyzed to determine theconcentration that inhibited end point by 50 percent (TC50).

For example, induction of cytochrome P450 enzymes in liver hepatocytesis a key factor that determines the efficacy and toxicity of drugs. Inparticular, induction of P450s is an important mechanism of troublesomedrug-drug interactions, and it is also an important factor that limitsdrug efficacy and governs drug toxicity. Cytochrome P450 inductionassays have been difficult to develop, because they require intactnormal human hepatocytes. These cells have proven intractable toproduction in numbers sufficient to sustain mass production of highthroughput assays.

For example, according to this aspect of the invention, a candidatecompound may be contacted with cell or organoid as described herein, andany change to the cells or in to activity of the cells may be monitored.Examples of other non-therapeutic uses of the cells or organoids of thepresent invention include research of liver embryology, liver celllineages, and differentiation pathways; gene expression studiesincluding recombinant gene expression; mechanisms involved in liverinjury and repair; research of inflammatory and infectious diseases ofthe liver; studies of pathogenetic mechanisms; and studies of mechanismsof liver cell transformation and aetiology of liver cancer.

For high-throughput purposes, said liver organoids are cultured inmultiwell plates such as, for example, 96 well plates or 384 wellplates. Libraries of molecules are used to identify a molecule thataffects said organoids. Preferred libraries comprise antibody fragmentlibraries, peptide phage display libraries, peptide libraries (e.g.LOPAP™, Sigma Aldrich), lipid libraries (BioMol), synthetic compoundlibraries (e.g. LOP AC™, Sigma Aldrich) or natural compound libraries(Specs, TimTec). Furthermore, genetic libraries can be used that induceor repress the expression of one of more genes in the progeny of theadenoma cells. These genetic libraries comprise cDNA libraries,antisense libraries, and siRNA or other non-coding RNA libraries. Thecells are preferably exposed to multiple concentrations of a test agentfor certain period of time. At the end of the exposure period, thecultures are evaluated. The term “affecting” is used to cover any changein a cell, including, but not limited to, a reduction in, or loss of,proliferation, a morphological change, and cell death. Said liverorganoids can also be used to identify drugs that specifically targetepithelial carcinoma cells, but not said liver organoids.

Liver organoids according to the invention can further replace the useof cell lines such as Caco-2 cells in toxicity assays of potential noveldrugs or of known or novel food supplements.

Furthermore, such liver organoids can be used for culturing of apathogen.

Cultures comprising liver organoids are useful in regenerative medicine,for example in post-radiation and/or post-surgery repair of the liverepithelium, in the repair of the epithelium in patients suffering fromchronic or acute liver failure or disease. Liver diseases include, butare not limited to Hepatocellular Carcinoma, Alagille Syndrome,Alpha-1-Antitrypsin Deficiency, Autoimmune Hepatitis, Biliary Atresia,Chronic Hepatitis, Cancer of the Liver, Cirrhosis Liver Cysts FattyLiver, Galactosemia Gilbert's Syndrome, Primary Biliary Cirrhosis,Hepatitis A, Hepatitis B, Hepatitis C, Primary Sclerosing Cholangitis,Reye's Syndrome, Sarcoidosis, Tyrosinemia, Type I Glycogen StorageDisease, Wilson's Disease, Neonatal Hepatitis, Non-alchoholicSteatoHepatitis, Porphyria, and Hemochromatosis.

Genetic conditions that lead to liver failure could benefit fromcell-based therapy in the form of partial or full cell replacement usingcells cultured according to the media and/or methods of the invention. Anon-limiting list of genetic conditions that lead to liver failureincludes: Progressive familial intrahepatic cholestasis, Glycogenstorage disease type III, Tyrosinemia, Deoxyguanosine kinase deficiency,Pyruvate carboxylase deficiency, Congenital dyserythropoietic anemia,Polycystic Liver Disease Polycystic Kidney Disease, Alpha-1 antitrypsinedeficiency, Ureum cycle defects, Organic acidemiea, lysosomal storagediseases, and Fatty Acid Oxydation Disorders. Other conditions that mayalso benefit from cell-based therapy include Wilson's Disease andHereditary Amyloidosis (FAP).

Other non-hepatocyte related causes of liver failure that would requirea full liver transplant to reach full therapeutic effect, may stillbenefit from some temporary restoration of function using cell-basedtherapy using cells cultured according to the media and/or methods ofthe invention. A non-limiting list of examples of such conditionsincludes: Primary Biliary Cirrhosis, Primary Sclerosing Cholangitis,Aglagille syndrome, Homozygous Familial hypercholesterolemia, HepatitisB with cirrhosis, Hepatitis C with cirrhosis, Budd-Chiari syndrome,Primary hyperoxaluria, Autoimmune Hepatitis, and Alcoholic liverdisease.

The liver organoids of the invention may be used in a method of treatinga hereditary disease that involves malfunctioning hepatocytes. Suchdiseases may be early onset or late onset. Early onset disease includemetabolite related organ failure (e.g. alpha-1-antitrypsin deficiency),glycogen storage diseases (e.g. GSD II, Pompe's disease), tyrosinemia,mild DGUOK, CDA type I, Ureum cycle defects (e.g. OTC deficiency),organic academia and fatty acid oxidation disorders. Late onset diseasesinclude primary hyperoxaluria, familial hypercholesterolemia, Wilson'sdisease, Hereditary Amyloidosis and Polycystic liver disease. Partial orfull replacement with healthy hepatocytes arising from liver organoidsof the invention may be used to restore liver function or to postponeliver failure.

The liver organoids of the invention may be used in a method of treatingchronic liver failure arising due to hereditary metabolic disease or asa result of hepatocyte infection. Treatment of a hereditary metabolicdisease may involve administration of genetically modified autologousliver organoids of the invention. Treatment of hepatocyte infections mayinvolve administration of allogeneic liver organoids of the invention.In some embodiments, the liver organoids are administered over a periodof 2-3 months.

The liver organoids of the invention may be used to treat acute liverfailure, for example, as a result of liver intoxication which may resultfrom use of paracetamol, medication or alcohol. In some embodiments, thetherapy to restore liver function will comprise injecting hepatocytesuspension from frozen, ready to use allogenic hepatocytes obtained fromorganoids of the invention. The ability to freeze suitable organoidsmeans that the organoids can be available for immediate delivery and soit is not necessary to wait for a blood transfusion.

In the case of replacement or correction of deficient liver function, itmay be possible to construct a cell-matrix structure from one or moreliver organoids generated according to the present invention. It isthought that only about 10% of hepatic cell mass is necessary foradequate function. This makes implantation of organoid unit compositionsinto children especially preferable to whole organ transplantation, dueto the relatively limited availability of donors and smaller size ofjuvenile organs. For example, an 8-month-old child has a normal liverthat weighs approximately 250 g. That child would therefore need about25 g of tissue. An adult liver weighs-approximately 1500 g; therefore,the required implant would only be about 1.5% of the adult liver. Whenorganoid units according to the invention are implanted, optionallyattached to a polymer scaffold, proliferation in the new host willoccur, and the resulting hepatic cell mass replaces the deficient hostfunction. The inventors have shown, for the first time, that it ispossible to generate mature hepatocytes from adult liver stem cells orliver tissue fragments comprising stem cells that are suitable fortransplantation into non-human animals or humans. Using the firstculture medium according to the invention, the inventors havedemonstrated that it is possible to maintain and expand a population ofliver stem cells. Using the second culture medium according to theinvention, the inventors have shown that hepatoblasts can bedifferentiated in vivo to mature hepatocytes suitable fortransplantation purposes. Hence, the inventors provide a new source ofhepatocytes for liver regeneration, replacement or correction ofdeficient liver function.

The inventors have also demonstrated successful transplantation of theorganoids, grown by methods of the present invention, intoimmunodeficient mice (see example 7), with transplanted organoid-derivedcells generating both cholangyocytes and hepatocytes in vivo. Therefore,in one embodiment the invention provides organoids or organoid-derivedcells of the invention for transplanting into human or animals.

The use of human liver organoids for transplantation purposes isadvantageous over the use of fetal or adult hepatocytes for a number ofreasons. Firstly, the culture methods of the invention provide unlimitedexpansion of cells and hence, an unlimited supply. In particular, theinventors have shown that under the correct culture conditions (e.g.using the expansion culture medium of the invention), that Lgr5+ cellscan undergo more than 1000 divisions in vitro. Therefore, Lgr5+ cellscan be extracted from the liver organoids and repassaged providing acontinual self-renewing source of transplantable hepatocyte andcholangyocyte-generating cells. By contrast, fetal or adult hepatocytesare derived from donor livers which only provide a single round oftransplantation. Furthermore, donor cells can only be kept alive for afew days but lose their hepatocyte properties. This means thetransplants must be made as soon as the donor becomes available.Organoid-derived cells, on the other hand, retain their phenotype overmultiple divisions and over prolonged periods of time meaning that theyare ready and available for transplantation at any stage. This couldalso allow the organoid-derived cells to be used as a temporary livertreatment to extend the lifespan of patients for patients on the waitinglist for liver transplants. A further advantage of the liver organoidsof the invention is that they can be frozen and later be defrostedwithout loss of function. This enables cell banking, easy storage andrapid availability for acute use. This could be useful for example, inthe preparation of an “off-the-shelf” product that might be used for thetreatment of acute liver toxicity. Organoids can also be grown fromcells or tissue fragments taken as small biopsies from live donorsminimising any ethical objections to the treatment. The donor may evenbe from the patient that is to be treated, which could reduce anynegative side-effects associated with transplantation of foreign cellsand organs and reduce the need for immunosuppressive drugs.

Accordingly, included within the scope of the invention are methods oftreatment of a human or animal patient through cellular therapy. Theterm “animal” here denotes all mammalian animals, preferably humanpatients. It also includes an individual animal in all stages ofdevelopment, including embryonic and foetal stages. For example, thepatient may be an adult, or the therapy may be for pediatric use (e.g.newborn, child or adolescent). Such cellular therapy encompasses theadministration of cells or organoids generated according to theinvention to a patient through any appropriate means. Specifically, suchmethods of treatment involve the regeneration of damaged tissue. Theterm “administration” as used herein refers to well recognized forms ofadministration, such as intravenous or injection, as well as toadministration by transplantation, for example transplantation bysurgery, grafting or transplantation of tissue engineered liver derivedfrom cells or organoids according to the present invention. In the caseof cells, systemic administration to an individual may be possible, forexample, by infusion into the superior mesenteric artery, the celiacartery, the subclavian vein via the thoracic duct, infusion into theheart via the superior vena cava, or infusion into the peritoneal cavitywith subsequent migration of cells via subdiaphragmatic lymphatics, ordirectly into liver sites via infusion into the hepatic arterial bloodsupply or into the portal vein.

Between 10⁴ and 10¹³ cells per 100 kg person may be administered perinfusion. Preferably, between about 1-5×10⁴ and 1-5×10⁷ cells may beinfused intravenously per 100 kg person. More preferably, between about1×10⁴ and 10×10⁶ cells may be infused intravenously per 100 kg person.In some embodiments, a single administration of cells or organoids isprovided. In other embodiments, multiple administrations are used.Multiple administrations can be provided over an initial treatmentregime, for example, of 3-7 consecutive days, and then repeated at othertimes.

In some embodiments it is desirable to repopulate/replace 10-20% of apatient's liver with healthy hepatocytes arising from a liver organoidof the invention.

It is also possible to reconstitute a liver organoid from one singlecell expressing Lgr5 as defined herein. This single cell may have beenmodified by introduction of a nucleic acid construct as defined herein,for example, to correct a genetic deficiency or mutation. It would alsobe possible to specifically ablate expression, as desired, for example,using siRNA. Potential polypeptides to be expressed could be any ofthose that are deficient in metabolic liver diseases, including, forexample, AAT (alpha antitrypsin). For elucidating liver physiology, wemight also express or inactivate genes implicated in the Wnt, EGF, FGF,BMP or notch pathway. Also, for screening of drug toxicity, theexpression or inactivation of genes responsible for liver drugmetabolism (for example, genes in the CYP family) would be of highinterest

In one embodiment, the expanded epithelial stem cells may bereprogrammed into related tissue fates such as, for example, liver cellsincluding a hepatocyte and a cholangiocyte cell. Thus far, it has notbeen possible to regenerate liver cells from adult stem cells. Theculturing methods of the present invention will enable to analyse forfactors that trans-differentiate the closely related epithelial stemcell to a liver cell, including a hepatocyte and a cholangiocyte cell.

It will be clear to a skilled person that gene therapy can additionallybe used in a method directed at repairing damaged or diseased tissue.Use can, for example, be made of an adenoviral or retroviral genedelivery vehicle to deliver genetic information, like DNA and/or RNA tostem cells. A skilled person can replace or repair particular genestargeted in gene therapy. For example, a normal gene may be insertedinto a nonspecific location within the genome to replace a nonfunctional gene. In another example, an abnormal gene sequence can bereplaced for a normal gene sequence through homologous recombination.Alternatively, selective reverse mutation can return a gene to itsnormal function. A further example is altering the regulation (thedegree to which a gene is turned on or off) of a particular gene.Preferably, the stem cells are ex vivo treated by a gene therapyapproach and are subsequently transferred to the mammal, preferably ahuman being in need of treatment. For example, organoid-derived cellsmay be genetically modified in culture before transplantation intopatients.

The inventors have found that Lgr5 is not detectable in healthy liver,although residual Lgr5 may be detected. Thus, the invention furtherprovides a method of diagnosing liver injury comprising detectingwhether Lgr5 is expressed, wherein the expression of Lgr5 proteinindicates liver injury. The invention also provides a method ofmonitoring the repair or regeneration of the liver by monitoring theexpression of Lgr5 in the liver. Lgr5 expression may be detected by anysuitable method, for example, flow cytometry, immunohistochemistry or byuse of PCR methods.

The invention also provides a composition or cell culture vesselcomprising cells and/or organoids according to any one of the aspects ofthe invention described above, and a culture medium according to any oneof the aspects of the invention described above. For example, such acomposition or cell culture vessel may comprise any number of cells ororganoids cultured according to a method of the invention, in a culturemedium as described above. For example, a preferred culture mediumcomprises or consists of Advanced-DMEM/F12 supplemented with B27, N2,200 ng/ml N-Acetylcysteine, 50 ng/ml EGF, 1 μg/ml R-spondin 1, 10 nMgastrin, 100 ng/ml FGF10, 10 mM Nicotinamide and 50 ng/ml HGF and 50%Wnt conditioned media.

Definitions

A nucleic acid construct comprises a nucleic acid molecule of interestand will ensure expression of the given nucleic acid molecule in thecells wherein it had been introduced. A particularly preferred nucleicacid construct is an expression vector wherein a nucleic acid moleculeencoding a polypeptide is operably linked to a promoter capable ofdirecting expression of said nucleic acid molecule (i.e a codingsequence) in said cells. The phrase “expression vector” generally refersto a nucleic acid molecule that is capable of effecting expression of agene/nucleic acid molecule it contains in a cell compatible with suchsequences. These expression vectors typically include at least suitablepromoter sequences and optionally, transcription termination signals. Anucleic acid or DNA or nucleotide sequence encoding a polypeptide isincorporated into a DNA/nucleic acid construct capable of introductioninto and expression in an in vitro cell culture as identified in amethod of the invention. A DNA construct prepared for introduction intoa particular cell typically include a replication system recognized bysaid cell, an intended DNA segment encoding a desired polypeptide, andtranscriptional and translational initiation and termination regulatorysequences operably linked to the polypeptide-encoding segment. A DNAsegment is “operably linked” when it is placed into a functionalrelationship with another DNA segment. For example, a promoter orenhancer is operably linked to a coding sequence if it stimulates thetranscription of the sequence. DNA for a signal sequence is operablylinked to DNA encoding a polypeptide if it is expressed as a preproteinthat participates in the secretion of a polypeptide. Generally, a DNAsequence that is operably linked are contiguous, and, in the case of asignal sequence, both contiguous and in reading phase. However,enhancers need not be contiguous with a coding sequence whosetranscription they control. Linking is accomplished by ligation atconvenient restriction sites or at adapters or linkers inserted in lieuthereof.

The selection of an appropriate promoter sequence generally depends uponthe host cell selected for the expression of a DNA segment. Examples ofsuitable promoter sequences include eukaryotic promoters well known inthe art (see, e.g. Sambrook and Russell, 2001, supra). A transcriptionalregulatory sequence typically includes a heterologous enhancer orpromoter that is recognised by the cell. Suitable promoters include theCMV promoter. An expression vector includes the replication system andtranscriptional and translational regulatory sequences together with theinsertion site for the polypeptide encoding segment can be employed.Examples of workable combinations of cell lines and expression vectorsare described in Sambrook and Russell (2001, supra) and in Metzger etal. (1988) Nature 334: 31-36.

The invention is not limited to a specific polypeptide or nucleic acidmolecule of interest to be expressed in a cell expressing Lgr5 asidentified herein. Depending on the aim of the method, one may envisageto express a polypeptide in a liver cell and/or to inactivate theexpression of a polypeptide in a liver cell. Potential polypeptides tobe expressed could be all of those that are deficient in metabolic liverdiseases, as eg AAT (alpha antitrypsin). For elucidating the liverphysiology, we might also express or inactivate genes implicated in theWnt, EGF, FGF, BMP or notch pathway. Also, for screening of drugtoxicity, the expression or inactivation of genes responsible for liverdrug metabolism (e.g. genes of the CYP family) would be of highinterest.

Some aspects of the invention concern the use of a nucleic acidconstruct or expression vector comprising a nucleotide sequence asdefined above, wherein the vector is a vector that is suitable for genetherapy. Vectors that are suitable for gene therapy are described inAnderson 1998, Nature 392: 25-30; Walther and Stein, 2000, Drugs 60:249-71; Kay et al., 2001, Nat. Med. 7: 33-40; Russell, 2000, J. Gen.Virol. 81: 2573-604; Amado and Chen, 1999, Science 285: 674-6; Federico,1999, Curr. Opin. Biotechnol. 10: 448-53; Vigna and Naldini, 2000, J.Gene Med. 2: 308-16; Marin et al., 1997, Mol. Med. Today 3: 396-403;Peng and Russell, 1999, Curr. Opin. Biotechnol. 10: 454-7; Sommerfelt,1999, J. Gen. Virol. 80: 3049-64; Reiser, 2000, Gene Ther. 7: 910-3; andreferences cited therein. Examples include integrative andnon-integrative vectors such as those based on retroviruses,adenoviruses (AdV), adeno-associated viruses (AAV), lentiviruses, poxviruses, alphaviruses, and herpes viruses.

A particularly suitable gene therapy vector includes an Adenoviral (Ad)and Adeno-associated virus (AAV) vector. These vectors infect a widenumber of dividing and non-dividing cell types including liver cells. Inaddition adenoviral vectors are capable of high levels of transgeneexpression. However, because of the episomal nature of the adenoviraland AAV vectors after cell entry, these viral vectors are most suitedfor therapeutic applications requiring only transient expression of thetransgene (Russell, 2000, J. Gen. Virol. 81: 2573-2604; Goncalves, 2005,Virol J. 2(1):43) as indicated above. Preferred adenoviral vectors aremodified to reduce the host response as reviewed by Russell (2000,supra). Safety and efficacy of AAV gene transfer has been extensivelystudied in humans with encouraging results in the liver, muscle, CNS,and retina (Manno et al Nat medicine 2006, Stroes et al ATVB 2008,Kaplitt, Feigin, Lancet 2009; Maguire, Simonelli et al NEJM 2008;Bainbridge et al NEJM 2008). AAV2 is the best characterized serotype forgene transfer studies both in humans and experimental models. AAV2presents natural tropism towards skeletal muscles, neurons, vascularsmooth muscle cells and hepatocytes. AAV2 is therefore a good choice ofvector to target liver tissues. Other examples of adeno-associatedvirus-based non integrative vectors include AAV1, AAV3, AAV4, AAV5,AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and pseudotyped AAV. The use ofnon-human serotypes, like AAV8 and AAV9, might be useful to overcomethese immunological responses in subjects, and clinical trials have justcommenced (ClinicalTrials.gov Identifier: NCT00979238). For genetransfer into a liver cell, an adenovirus serotype 5 or an AAV serotype2, 7 or 8 have been shown to be effective vectors and therefore apreferred Ad or AAV serotype (Gao, Molecular Therapy (2006) 13, 77-87).

A preferred retroviral vector for application in the present inventionis a lentiviral based expression construct. Lentiviral vectors have theunique ability to infect non-dividing cells (Amado and Chen, 1999Science 285: 674-6). Methods for the construction and use of lentiviralbased expression constructs are described in U.S. Pat. Nos. 6,165,782,6,207,455, 6,218,181, 6,277,633 and 6,323,031 and in Federico (1999,Curr Opin Biotechnol 10: 448-53) and Vigna et al. (2000, J Gene Med2000; 2: 308-16).

Generally, gene therapy vectors will be as the expression vectorsdescribed above in the sense that they comprise a nucleotide sequenceencoding a polypeptide of the invention to be expressed, whereby anucleotide sequence is operably linked to the appropriate regulatorysequences as indicated above. Such regulatory sequence will at leastcomprise a promoter sequence. Suitable promoters for expression of anucleotide sequence encoding a polypeptide from gene therapy vectorsinclude e.g. cytomegalovirus (CMV) intermediate early promoter, virallong terminal repeat promoters (LTRs), such as those from murine moloneyleukaemia virus (MMLV) rous sarcoma virus, or HTLV-1, the simian virus40 (SV 40) early promoter and the herpes simplex virus thymidine kinasepromoter. Suitable promoters are described below.

Several inducible promoter systems have been described that may beinduced by the administration of small organic or inorganic compounds.Such inducible promoters include those controlled by heavy metals, suchas the metallothionine promoter (Brinster et al. 1982 Nature 296: 39-42;Mayo et al. 1982 Cell 29: 99-108), RU-486 (a progesterone antagonist)(Wang et al. 1994 Proc. Natl. Acad. Sci. USA 91: 8180-8184), steroids(Mader and White, 1993 Proc. Natl. Acad. Sci. USA 90: 5603-5607),tetracycline (Gossen and Bujard 1992 Proc. Natl. Acad. Sci. USA 89:5547-5551; U.S. Pat. No. 5,464,758; Furth et al. 1994 Proc. Natl. Acad.Sci. USA 91: 9302-9306; Howe et al. 1995 J. Biol. Chem. 270:14168-14174; Resnitzky et al. 1994 Mol. Cell. Biol. 14: 1669-1679;Shockett et al. 1995 Proc. Natl. Acad. Sci. USA 92: 6522-6526) and thetTAER system that is based on the multi-chimeric transactivator composedof a tetR polypeptide, as activation domain of VP16, and a ligandbinding domain of an estrogen receptor (Yee et al., 2002, U.S. Pat. No.6,432,705).

Suitable promoters for nucleotide sequences encoding small RNAs forknock down of specific genes by RNA interference (see below) include, inaddition to the above mentioned polymerase II promoters, polymerase IIIpromoters. The RNA polymerase III (pol III) is responsible for thesynthesis of a large variety of small nuclear and cytoplasmic non-codingRNAs including 5S, U6, adenovirus VA1, Vault, telomerase RNA, and tRNAs.The promoter structures of a large number of genes encoding these RNAshave been determined and it has been found that RNA pol III promotersfall into three types of structures (for a review see Geiduschek andTocchini-Valentini, 1988 Annu. Rev. Biochem. 57: 873-914; Willis, 1993Eur. J. Biochem. 212: 1-11; Hernandez, 2001, J. Biol. Chem. 276:26733-36). Particularly suitable for expression of siRNAs are the type 3of the RNA pol III promoters, whereby transcription is driven bycis-acting elements found only in the 5′-flanking region, i.e. upstreamof the transcription start site. Upstream sequence elements include atraditional TATA box (Mattaj et al., 1988 Cell 55, 435-442), proximalsequence element and a distal sequence element (DSE; Gupta and Reddy,1991 Nucleic Acids Res. 19, 2073-2075). Examples of genes under thecontrol of the type 3 pol III promoter are U6 small nuclear RNA (U6snRNA), 7SK, Y, MRP, H1 and telomerase RNA genes (see e.g. Myslinski etal., 2001, Nucl. Acids Res. 21: 2502-09).

A gene therapy vector may optionally comprise a second or one or morefurther nucleotide sequence coding for a second or further polypeptide.A second or further polypeptide may be a (selectable) marker polypeptidethat allows for the identification, selection and/or screening for cellscontaining the expression construct. Suitable marker proteins for thispurpose are e.g. the fluorescent protein GFP, and the selectable markergenes HSV thymidine kinase (for selection on HAT medium), bacterialhygromycin B phosphotransferase (for selection on hygromycin B), Tn5aminoglycoside phosphotransferase (for selection on G418), anddihydrofolate reductase (DHFR) (for selection on methotrexate), CD20,the low affinity nerve growth factor gene. Sources for obtaining thesemarker genes and methods for their use are provided in Sambrook andRussel (2001) “Molecular Cloning: A Laboratory Manual (3^(rd) edition),Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, NewYork.

Alternatively, a second or further nucleotide sequence may encode apolypeptide that provides for fail-safe mechanism that allows a subjectfrom the transgenic cells to be cured, if deemed necessary. Such anucleotide sequence, often referred to as a suicide gene, encodes apolypeptide that is capable of converting a prodrug into a toxicsubstance that is capable of killing the transgenic cells in which thepolypeptide is expressed. Suitable examples of such suicide genesinclude e.g. the E. coli cytosine deaminase gene or one of the thymidinekinase genes from Herpes Simplex Virus, Cytomegalovirus andVaricella-Zoster virus, in which case ganciclovir may be used as prodrugto kill the IL-10 transgenic cells in the subject (see e.g. Clair etal., 1987, Antimicrob. Agents Chemother. 31: 844-849).

For knock down of expression of a specific polypeptide, a gene therapyvector or other expression construct is used for the expression of adesired nucleotide sequence that preferably encodes an RNAi agent, i.e.an RNA molecule that is capable of RNA interference or that is part ofan RNA molecule that is capable of RNA interference. Such RNA moleculesare referred to as siRNA (short interfering RNA, including e.g. a shorthairpin RNA). Alternatively, a siRNA molecule may directly, e.g. in apharmaceutical composition that is administered within or in theneighborhood of a cell expressing Lgr5, or of a liver cell (i.e.hepatocyte or cholangiocyte) or of a liver organoid.

A desired nucleotide sequence comprises an antisense code DNA coding forthe antisense RNA directed against a region of the target gene mRNA,and/or a sense code DNA coding for the sense RNA directed against thesame region of the target gene mRNA. In a DNA construct of theinvention, an antisense and sense code DNAs are operably linked to oneor more promoters as herein defined above that are capable of expressingan antisense and sense RNAs, respectively. “siRNA” preferably means asmall interfering RNA that is a short-length double-stranded RNA thatare not toxic in mammalian cells (Elbashir et al., 2001, Nature 411:494-98; Caplen et al., 2001, Proc. Natl. Acad. Sci. USA 98: 9742-47).The length is not necessarily limited to 21 to 23 nucleotides. There isno particular limitation in the length of siRNA as long as it does notshow toxicity. “siRNAs” can be, e.g. at least 15, 18 or 21 nucleotidesand up to 25, 30, 35 or 49 nucleotides long. Alternatively, thedouble-stranded RNA portion of a final transcription product of siRNA tobe expressed can be, e.g. at least 15, 18 or 21 nucleotides and up to25, 30, 35 or 49 nucleotides long.

“Antisense RNA” is preferably an RNA strand having a sequencecomplementary to a target gene mRNA, and thought to induce RNAi bybinding to the target gene mRNA. “Sense RNA” has a sequencecomplementary to the antisense RNA, and annealed to its complementaryantisense RNA to form siRNA. The term “target gene” in this contextpreferably refers to a gene whose expression is to be silenced due tosiRNA to be expressed by the present system, and can be arbitrarilyselected. As this target gene, for example, genes whose sequences areknown but whose functions remain to be elucidated, and genes whoseexpressions are thought to be causative of diseases are preferablyselected. A target gene may be one whose genome sequence has not beenfully elucidated, as long as a partial sequence of mRNA of the genehaving at least 15 nucleotides or more, which is a length capable ofbinding to one of the strands (antisense RNA strand) of siRNA, has beendetermined. Therefore, genes, expressed sequence tags (ESTs) andportions of mRNA, of which some sequence (preferably at least 15nucleotides) has been elucidated, may be selected as the “target gene”even if their full length sequences have not been determined.

The double-stranded RNA portions of siRNAs in which two RNA strands pairup are not limited to the completely paired ones, and may containnonpairing portions due to mismatch (the corresponding nucleotides arenot complementary), bulge (lacking in the corresponding complementarynucleotide on one strand), and the like. A non-pairing portions can becontained to the extent that they do not interfere with siRNA formation.The “bulge” used herein preferably comprise 1 to 2 non-pairingnucleotides, and the double-stranded RNA region of siRNAs in which twoRNA strands pair up contains preferably 1 to 7, more preferably 1 to 5bulges. In addition, the “mismatch” used herein is preferably containedin the double-stranded RNA region of siRNAs in which two RNA strandspair up, preferably 1 to 7, more preferably 1 to 5, in number. In apreferable mismatch, one of the nucleotides is guanine, and the other isuracil. Such a mismatch is due to a mutation from C to T, G to A, ormixtures thereof in DNA coding for sense RNA, but not particularlylimited to them. Furthermore, in the present invention, adouble-stranded RNA region of siRNAs in which two RNA strands pair upmay contain both bulge and mismatched, which sum up to, preferably 1 to7, more preferably 1 to 5 in number. Such non-pairing portions(mismatches or bulges, etc.) can suppress the below-describedrecombination between antisense and sense code DNAs and make the siRNAexpression system as described below stable. Furthermore, although it isdifficult to sequence stem loop DNA containing no non-pairing portion inthe double-stranded RNA region of siRNAs in which two RNA strands pairup, the sequencing is enabled by introducing mismatches or bulges asdescribed above. Moreover, siRNAs containing mismatches or bulges in thepairing double-stranded RNA region have the advantage of being stable inE. coli or animal cells.

The terminal structure of siRNA may be either blunt or cohesive(overhanging) as long as siRNA enables to silence the target geneexpression due to its RNAi effect. The cohesive (overhanging) endstructure is not limited only to the 3′ overhang, and the 5′ overhangingstructure may be included as long as it is capable of inducing the RNAieffect. In addition, the number of overhanging nucleotide is not limitedto the already reported 2 or 3, but can be any numbers as long as theoverhang is capable of inducing the RNAi effect. For example, theoverhang consists of 1 to 8, preferably 2 to 4 nucleotides. Herein, thetotal length of siRNA having cohesive end structure is expressed as thesum of the length of the paired double-stranded portion and that of apair comprising overhanging single-strands at both ends. For example, inthe case of 19 bp double-stranded RNA portion with 4 nucleotideoverhangs at both ends, the total length is expressed as 23 bp.Furthermore, since this overhanging sequence has low specificity to atarget gene, it is not necessarily complementary (antisense) oridentical (sense) to the target gene sequence. Furthermore, as long assiRNA is able to maintain its gene silencing effect on the target gene,siRNA may contain a low molecular weight RNA (which may be a natural RNAmolecule such as tRNA, rRNA or viral RNA, or an artificial RNAmolecule), for example, in the overhanging portion at its one end.

In addition, the terminal structure of the “siRNA” is necessarily thecut off structure at both ends as described above, and may have astem-loop structure in which ends of one side of double-stranded RNA areconnected by a linker RNA (a “shRNA”). The length of the double-strandedRNA region (stem-loop portion) can be, e.g. at least 15, 18 or 21nucleotides and up to 25, 30, 35 or 49 nucleotides long. Alternatively,the length of the double-stranded RNA region that is a finaltranscription product of siRNAs to be expressed is, e.g. at least 15, 18or 21 nucleotides and up to 25, 30, 35 or 49 nucleotides long.Furthermore, there is no particular limitation in the length of thelinker as long as it has a length so as not to hinder the pairing of thestem portion. For example, for stable pairing of the stem portion andsuppression of the recombination between DNAs coding for the portion,the linker portion may have a clover-leaf tRNA structure. Even thoughthe linker has a length that hinders pairing of the stem portion, it ispossible, for example, to construct the linker portion to includeintrons so that the introns are excised during processing of precursorRNA into mature RNA, thereby allowing pairing of the stem portion. Inthe case of a stem-loop siRNA, either end (head or tail) of RNA with noloop structure may have a low molecular weight RNA. As described above,this low molecular weight RNA may be a natural RNA molecule such astRNA, rRNA, snRNA or viral RNA, or an artificial RNA molecule.

To express antisense and sense RNAs from the antisense and sense codeDNAs respectively, a DNA construct of the present invention comprise apromoter as defined above. The number and the location of the promoterin the construct can in principle be arbitrarily selected as long as itis capable of expressing antisense and sense code DNAs. As a simpleexample of a DNA construct of the invention, a tandem expression systemcan be formed, in which a promoter is located upstream of both antisenseand sense code DNAs. This tandem expression system is capable ofproducing siRNAs having the aforementioned cut off structure on bothends. In the stem-loop siRNA expression system (stem expression system),antisense and sense code DNAs are arranged in the opposite direction,and these DNAs are connected via a linker DNA to construct a unit. Apromoter is linked to one side of this unit to construct a stem-loopsiRNA expression system. Herein, there is no particular limitation inthe length and sequence of the linker DNA, which may have any length andsequence as long as its sequence is not the termination sequence, andits length and sequence do not hinder the stem portion pairing duringthe mature RNA production as described above. As an example, DNA codingfor the above-mentioned tRNA and such can be used as a linker DNA.

In both cases of tandem and stem-loop expression systems, the 5′ end maybe have a sequence capable of promoting the transcription from thepromoter. More specifically, in the case of tandem siRNA, the efficiencyof siRNA production may be improved by adding a sequence capable ofpromoting the transcription from the promoters at the 5′ ends ofantisense and sense code DNAs. In the case of stem-loop siRNA, such asequence can be added at the 5′ end of the above-described unit. Atranscript from such a sequence may be used in a state of being attachedto siRNA as long as the target gene silencing by siRNA is not hindered.If this state hinders the gene silencing, it is preferable to performtrimming of the transcript using a trimming means (for example, ribozymeas are known in the art). It will be clear to the skilled person that anantisense and sense RNAs may be expressed in the same vector or indifferent vectors. To avoid the addition of excess sequences downstreamof the sense and antisense RNAs, it is preferred to place a terminatorof transcription at the 3′ ends of the respective strands (strandscoding for antisense and sense RNAs). The terminator may be a sequenceof four or more consecutive adenine (A) nucleotides.

In this document and in its claims, the verb “to comprise” and itsconjugations is used in its non-limiting sense to mean that itemsfollowing the word are included, but items not specifically mentionedare not excluded. In addition the verb “to consist” may be replaced by“to consist essentially of” meaning that a product as defined herein maycomprise additional component(s) than the ones specifically identified,said additional component(s) not altering the unique characteristic ofthe invention. In addition a method as defined herein may compriseadditional step(s) than the ones specifically identified, saidadditional step(s) not altering the unique characteristic of theinvention. In addition, reference to an element by the indefinitearticle “a” or “an” does not exclude the possibility that more than oneof the element is present, unless the context clearly requires thatthere be one and only one of the elements. The indefinite article “a” or“an” thus usually means “at least one”. The word “about” or“approximately” when used in association with a numerical value (about10) preferably means that the value may be the given value of 10 more orless 1% of the value.

All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

DESCRIPTION OF FIGURES

FIG. 1A-D: Liver organoids growth factor requirement. (A) DIC images ofliver organoids maintained with EGF (E), R-spondin 1 (R), Noggin (N),Wnt3A conditioned media (W) or the combination of them, supplementedwith FGF10, HGF and Nicotinamide. (B) The number of organoids wascounted weekly and passaged when required. Results are shown as mean±SEMof 3 independent experiments. (C) gene expression analysis by RTPCR ofLgr5, Keratin 7 (K7) and Albumin (Alb) genes. (D) Isolated bibliaryducts growing into organoids. Differential interference contrast imagesfrom the corresponding days after seeding. Magnification 10× (days 0, 1,3 and 5). Days 15 on magnification 4×. Cultures were passage every 4-7days by mechanical dissociation. Cultures have been grown at least for 8months.

FIG. 2A-B: Morphology of liver organoids. (A) Upper panels: paraffinsection of a mouse liver showing the different domains (PT=portal triad,CV=central vein). Lower panels: Paraffin section of a liver organoidshowing different domains b (single layered epithelia) and h (stratifiedepithelia) (B) Right pannel: Ecadherin staining in the liver organoids.Two different domains can be identified. Domain b, formed by a singlelayered epithelia that resembles the bile duct structures in the liver.This bile duct domain is formed by highly polarized cells that showspositive staining for pancytokeratin (PCK) (lower panel). Left panelsshow the presence of a second domain within the liver organoids. This hdomain is formed by a stratified epithelia with non-polarized cells. Thecells are organized around a central lumen and express the hepatocytemarker Alb. Magnification 10×.

FIG. 3: Wnt signalling in the liver cultures. Lac Z expression wasdetected in cultures derived from Lgr5-LacZ or Axin2 LacZ mice. Nopositive staining was detected in liver cultures derived from a B16mice. Magnification 4×, inset 20×.

FIG. 4A-B: Expression of liver differentiating markers. (A)immunohistochemical and immunofluorescence analysis of the expression ofthe cholangiocyte marker keratin 7 (K7) and the hepatocyte markerskeratin 8 (K8) and albumin (Alb). (B) analysis of the gene expression ofhepatocyte markers: Albumin (Alb), transthyretrin (Ttr), Glutaminesynthetase (Glu1), glucose 6 phosphatase (G6P) and Cytocrome p450isoform 3A11 (CYP3A11); and cholangiocyte markers keratin 7 (K7) andKeratin 19 (K19).

FIG. 5A-D: Liver single cell cultures. (A) flow cytometry plotindicating the area of the sorted cells. (B) single cell growing intoorganoids at the time points indicated. Magnification 40× (day 1-3), 10×(day 16), 4× (day 21-on). (C & D) representative image of the colonyformation efficiency of a Lgr5GFP single sorted cells. 100 cells wereseeded in triplicate and colonies were counted 10 days later.

FIG. 6A-B: Microarray analysis of the liver cultures. Analysis of thegene expression profile of adult liver tissue and liver organoidcultures maintained for 1 month in the ER or ER media supplemented withNoggin (ENR) or with noggin and Wnt (ENRW). The genetic profile wascompared between the different samples and the genetic profile of Browadipose tissue (BAT) white adipose tissue (WAT), muscle and new bornliver. (A) hit map analysis showing that the cultures present a similarprofile to the adult liver but a different profile to non-liver relatedtissues as muscle and BAT and WAT, (B) List of hepatocyte markers andcholangyocyte markers in the different conditions.

FIG. 7A-B: Mouse liver organoid culture shows stable karyotyping afterlong-term culture.

A—DIC images of liver organoids maintained in EGF (E) and R-spondin 1(R), supplemented with FGF10, HGF and Nicotinamide (left figure, ER) ormaintained in the same combination supplemented with Noggin (N) andWnt3A conditioned media (W) (right figure, ENRW) for a period of 24months.

B—Karyotype analysis of mouse liver organoids after 8 months in culture.Normal chromosomal counts (n=40, left panel figure) and polyploidy, atypical hepatocyte feature, were found (n=80, right panel figure)

FIG. 8A-E: Supplemental factors FGF10, HGF and Nicotinamide; effect ongrowth and differentiation.

A—Diagram depicting the genes differentially expressed during the 3stages of liver development, from hepatoblast to mature hepatocyte.

B—Scheme showing the protocol used. Cultures were seeded in expansionmedium EM2 (ERFHNic: EGF (E) and R-spondin 1 (R), supplemented withFGF10, HGF and Nicotinamide; ERFHNic is indicated as ‘ER’ in FIG. 8B) 2days prior the experiment. Two days later, culture media was changed toeither EGF (E) alone or EGF supplemented with R-spondin 1 (ER) with orwithout additional supplements chosen from FGF10 (F) or HGF (H) orNicotinamide (Nic) or a combination of these at the concentrationsstated in the text. Five days later cultures were split and replated at1:4 ratio for each condition. Under these conditions, cultures have beensplit and replated every 7 days for a total period of 10 weeks

C—First day after first split in each of the culture conditions tested.Results shows that EGF and R-spondin 1 combined with FGF10 or HGF orNicotinamide or a combination of these are essential to achieve at least1 passage.

D—After long-term culture, the combination of ER supplemented with FNicor ER supplemented with FHNic, both result in high passage numbers.After passage 10, the growth rate is better for the culture conditionincluding the 3 supplemental factors; ERFHNic (FIG. 15 A, B).

E—RT-PCR analysis showing the expression of different hepatocyte markers(CYP3A11, Alb, FAH) and cholangiocyte marker (K19) and stem cell markerIGR5 5 days after the withdrawal of certain factors (starting point wasERFHNic). Note that only the condition EF showed expression of allhepatocyte markers tested. HPRT was used as a housekeeping gene tonormalize for gene expression.

FIG. 9: Table showing the quantification of different hepatocyte andcholangiocyte specific transcription factors in cells from threedifferent liver culture conditions and in adult liver tissue. Also shownis the expression of the key components of the Notch and TGF-betasignalling pathways. E=EFHNic, ER=ERFHNic, ENRW=ENRWFHNic.

FIG. 10A-F: Differentiation protocol

A—Scheme showing the protocol used. Cultures were seeded in expansionmedium (ERFHNic: EGF (E) and R-spondin 1 (R), supplemented with FGF10,HGF and Nicotinamide; ERFHNic is indicated as ‘ER’ in FIG. 10A) 2 daysprior to the experiment. Two days later, culture media was changed toEGF (E) supplemented with either A8301 (A), or DAPT (D), or FGF10 (F) orHGF (H) or Nicotinamide (Nic) or R-spondin 1 (R) or Wnt3A or Noggin (N)or a combination of these at the concentrations shown. RNA was isolatedat several time points. Mouse liver tissue was taken as positive control(+) whereas water was taken as negative control (−).

B—RT-PCR analysis showing the expression of the hepatocyte markersCYP3A11, Alb, FAH, tbx3, TAT and Gck 7 days after differentiationconditions. Note that only the condition EADF showed an expression ofall hepatocyte markers tested. HPRT was used as a housekeeping gene tonormalize for gene expression.

C—Time course expression analysis after differentiation conditions. Atdays 2, 5 and 8 days after differentiation, the expression of thehepatocyte markers CYP3A11, Alb, FAH, and the cholangyocyte marker K19,was analysed by RTPCR. Note that the expression of the liver markersCYP3A11 and FAH starts at day 5 and peaks at day 8 after. HPRT was usedas a housekeeping gene to normalize for gene expression. A; A8301, D;DAPT, F; FGF10, H; HGF, De; Dexamethasone

D—Titration experiment showing the expression of the hepatocyte markersCYP3A11, Alb, FAH, tbx3, TAT, G6P and Gck 7 days after differentconcentrations of the differentiation compounds A and D. HPRT was usedas a housekeeping gene to normalize for gene expression.

E—Immunofluorescent staining for the liver markers K19, Albumin andhepatocyte surface marker. Hoeschst was use to stain nuclei.

F—Xgal staining on Albcreert2LacZ mice liver-derived organoids. Albuminpositive cells (arrows) were detected after EADF differentiation intamoxifen induced Albcreert2LacZ derived cultures.

FIG. 11: Human-derived liver cultures under ERFHNic culture conditions

FIG. 12A-B: Liver response to Wnt signaling stimulation underphysiological conditions or during regeneration after injury

A: Injection of Lgr5 ligand R-spondin1 in Axin2 LacZ mice shows thatliver cells are responsive to Wnt stimulation (arrows pointing X-galpositive cells). There was no Lgr5 expression so the inventorshypothesise that Lgr4 was used to initiate the response.

B: CCL4 injection in Axin2 LAcZ mice shows that during the regenerationresponse Wnt signalling is activated

FIG. 13A-B: Lgr5 upregulation following liver injury-regeneration model.

Adult Lgr5-LacZ KI mice were injected with 0.8 ml/kg of the hepatotoxiccompound CCL4. The pictures show that in non injected (undamaged) liversthe Wnt pathway is active only in cells lining the ducts. After damageby CC14 cells also cells not lining duct have an activated Wnt pathway.

A—Time course experiment showing upregulation of Lgr5 in CCL4 damagedlivers (arrows showing x-gal positive cells). Control CCL4 injected WTmice and placebo-injected Lgr5LacZ Ki mice did not show any staining(right-hand panel).

B—Lgr5 co-staining with liver markers.

FIG. 14: Isolated duct staining for K19

Lgr5LacZ duct isolation. K19 staining confirms that the isolated andseeded structures are indeed intrahepatic ducts.

FIG. 15A-B: Growth factor requirement

The 3 supplemental factors (FGF10, HGF and Nicotinamide) are essentialfor long term self-maintenance of liver cultures. After long-termculture, the combination of ER including FNic ($) or ERFHNic ($$), bothresult in high passage numbers. After passage 10, the growth rate isbetter for the culture condition including the 3 supplemental factors;ERFHNic (see FIG. 16B).

FIG. 16A-C: Gene expression profile of mouse liver organoids underdifferentiation conditions resemble the adult and newborn liver profile

A—Gene clusters showing the genes similarly expressed (a) or similarlyshut down (b) between the differentiation condition EADF and adult ornewborn liver.

B—Gene clusters showing the genes differentially expressed between theliver organoids and adult or newborn liver (a) and the genes similarlyexpressed between EADF and newborn liver (b).

C—Raw signal data from a microarray analysis, comparing the expressionlevels of selected ductal markers, transcription factors necessary forNgn3 expression and endocrine markers in adult liver, adult pancreas,pancreas organoids and liver organoids in expansion media.

FIG. 17: Transplantation of the cells into mouse model of liver disease

Organoids were transplanted into the mouse model: adult FGR mice(FAH−/−RAG−/−IL2R−/−). Hepatocytes were transplanted into the mice as acontrol.

A—K19 positive cells (left top panel) and Fah positive cells (middlepanel) derived from the liver organoids transplanted into FAH knock outmice. Hepatocyte transplanted control (top right panel). Lower flowCytometry plots show that the % of hepatocyte positive cells was higherin the group that resulted in positive FAH engrafted hepatocytes.

B & C—Flow Cytometry analyses of cells transplanted. (C) Clone 1,obtained from Lgr5-GFP mouse, and (D) clone 2, obtained from Lgr5-lacZmouse. The hepatocyte surface marker shows a positive subpopulation thatcomprises large cells and highly granular cells, i.e. cells thatrepresent the phenotype of mature hepatocytes.

D—Transplantation schedule.

FIG. 18: Mouse liver signature genes

Table showing a) markers expressed in mouse liver stem cells; b) markersnot expressed in mouse liver stem cells; c) hepatocyte and cholangiocytemarkers expressed in mouse liver stem cell signature for mouse liverorganoids in expansion media; d) hepatocyte and cholangiocyte markersnot expressed in mouse liver stem cell signature for mouse liverorganoids in expansion media; e) reprogramming genes expressed in mouseliver organoids; f) reprogramming genes not expressed in mouse liverorganoids. The results were obtained using a liver microarray using theUniversal Mouse Reference RNA (Strategene, Catalog #740100) as areference RNA. If the absolute figures detected were less than 100, thegene was consider as undetected.

FIG. 19A-E: Human liver signature genes

Table showing results of liver mircroarray of human organoids. From leftto right, the results are shown for a) expansion medium EM1, b)expansion medium EM2, c) differentiation medium, d) adult liver.

The numbers (log 2) in the left four columns are the result of acomparison between the sample and a reference (commercial) RNA samplewhich is used for all arrays. The relative expression of mRNA in eachsample compared to the RNA present in the reference sample is shown. Thereference RNA used was Universal Human Reference RNA (Stratagene,Catalog #740000). Thus, negative numbers in these columns do not relateto real expression levels it just means there is less of that RNA thenin the Reference sample. The 4 columns on the right are absolutefigures. If they are below 100, they are considered as undetected.

EXAMPLES Example 1—an Expansion Medium for Liver Organoid Growth andExpansion

After isolation, biliary ducts (see FIG. 1) were suspended in MATRIGEL™and cultured in different growth factor conditions. The combination ofEGF (50 ng/ml) and R-spondin 1 (1 ug/ml) supplemented with FGF10 (100ng/ml), HGF (25-50 ng/ml) and Nicotinamide (1-10 mM), (ERFHNic) wereessential for the long term maintenance of the cultures, indicating thatWnt signalling and EGF signalling are strictly required to maintainadult liver progenitor proliferation in vitro. The addition of Noggin(100 ng/ml) and Wnt conditioned media (50%) also showed long termmaintenance of the cultures (see FIGS. 1A and 1B). Under theseconditions that supported long-term maintenance, Lgr5 expression as wellas hepatocyte markers (Albumin) and cholangiocyte markers (K7) weredetected by RT-PCR (see FIG. 1C). Under these conditions liver organoidshave been weekly passaged by mechanical or enzymatic dissociation, at1:8 dilution, and have been grown for many months (FIG. 1D).

We analysed the expression of the Wnt target genes Axin2 and Lgr5 in thecultures. Cultures of both Axin2LacZ and Lgr5-LacZ livers revealed thepresence of Axin2- and Lgr5-positive cells in the liver organoids 1month after seeding, thus confirming that the Wnt signalling is activeand required for culture growth (FIG. 3). The liver cultures alsoexpress hepatocyte markers (e.g. albumin, transthyretrin, Glutaminesynthetase) and cholangiocyte makers (Keratin 7 and 19) (see FIG. 4).

When single Lgr5 cells from a Lgr5LacZ or Lgr5GFP mouse were sorted,single colonies grew into organoids. These cultures also express markersof cholangiocyte and hepatocyte lineages and have been maintained andregularly split into 1:6-1:8 for more than 4 months (see FIGS. 5A & B).Interestingly, only the cultures derived from Lgr5 positive cells grewinto organoids (FIGS. 5C & D). These data indicate that Lgr5 cells areprogenitor cells of these cultures and able to propagate progeny of the2 different liver lineages.

Having established that the liver organoids are derived from Lgr5+vecells we set out to determine their individual gene signature ascompared to the adult liver signature. RNA was isolated from adult liverand from liver organoids grown in ER or ENRW media supplemented withFGF10, Nicotinamide and Hepatocyte Growth Factor. The genetic signatureof the adult liver and the 2 liver culture conditions was subsequentlyderived via comparative gene expression profiling in respect to theexpression of a Universal RNA reference. The use of the same referenceRNA for the hybridization to all the samples allowed us to compare the 3independent samples among them (adult liver, ER and ENRW). The heat mapanalysis revealed that the expression profile of both culture conditionshighly resemble the adult liver tissue expression profile, whereas theydo not share the same profile when compared to muscle or adipose tissueprofile (see FIG. 6.). Among the similar gene expression profile betweenthe adult liver and the liver cultures, liver specific genes as HNF1a,HNF1b, HNF4, Alb, Glu1, Met, G6P, Fahd1, Fahd2a, CYP4B1, K7 and K19 aredetected. The heat map analysis reveals that both culture conditionspresent similar expression pattern among each other and when compared tothe adult liver sample. However, when analyzing the data in detail, wecan observe that the condition without Wnt and without noggin shows amore differentiated pattern that the condition including both growthfactors. This is in agreement with the data shown in FIG. 1 C wherehepatocyte differentiation (by means of albumin expression) is almostabsent in the presence of Wnt. This result would indicate that Wnt isfavouring the self-renewal of the culture in detriment of thedifferentiation.

Also, in both culture conditions as well as in the adult liver,non-specific adult liver genes as AFP, and non-liver transcriptionfactors as Pdx1 or NeuroD can be detected.

It is remarkable that, in both culture conditions but not in the adultliver, the stem cell marker Lgr5 was one of the most highly enrichedgenes in the liver culture signature. Also, cell markers of progenitorpopulations in small intestine and stomach as Cd44 and Sox9 (Barker &Huch et al Cell stem cell 2010) were highly expressed in both cultureconditions but not in adult liver, indicating again the self-renewalcapacity of the liver cultures as well as the quiescent status of thenormal adult liver.

Additionally, apart from Lgr5, multiple Wnt target genes were alsohighly upregulated in the liver cultures compared to the adult liverincluding MMP7, Sp5 and Tnfrs19, among others, providing strong evidenceof the requirement of an active and robust canonical Wnt signalingactivity to maintain the self renewing capacity of the cultures.

Example 2—an Improved Differentiation Medium

Under ER or ENRW conditions the liver cultures self-renew, and can bemaintained and expanded in a weekly basis, for up to 1 year (FIG. 7A).The karyotypic analysis after 1 year shows no evidence of chromosomalaberrations. More than 66% of the cells analysed presented normalchromosomal counts and 13% of them also showed polyploidy, acharacteristic trait of hepatocytes (FIG. 7B).

The combination of EGF (50 ng/ml) and R-spondin 1 (1 ug/nil)supplemented with FGF10 (100 ng/ml), HGF (25-50 ng/ml) and Nicotinamide(1-10 mM), were preferable for the long term maintenance of thecultures. Under these conditions, we obtained long-lived cell culturesthat express biliary duct and some hepatoblast or immature-hepatocytemarkers (Glu1, Albumine). However, the number of cells positive forthese hepatocyte markers was very low. Under these culture conditions,no mature hepatocyte markers (e.g. p450 Cytochromes) were detected.These results suggest that the culture conditions described herefacilitate the expansion of liver progenitors able to generatehepatocyte-like cells, albeit at lower numbers, but not fully maturehepatocytes (FIG. 8A).

To enhance the hepatocytic nature of the cultures and obtain maturehepatocytes in vitro, we first determined whether the three supplementalfactors (FGF10, HGF and Nicotinamide) added to EGF and R-spondin1 wereexerting either a positive or negative effect on the hepatocyteexpression, as well as on the self-renewal of the culture. We generatedliver organoid cultures and cultured them either with EGF or EGF andR-spondin1 plus FGF10 or HGF or Nicotinamide or the combination ofthese, and we split the cultures once a week for a total period of 10weeks. At each time-point we also analysed the expression of severalmature hepatocyte markers (FAH, CYP3A11) and hepatoblast markers(albumin) (FIG. 8B).

In agreement with the data in FIG. 1 (see example 1), we observed thatR-spondin1 and Nicotinamide combined with FGF10 are essential for thegrowth and self-renewal of the liver cultures (FIGS. 8C&D). R-spondin1and Nicotinamide both inhibit the expression of the mature markerCYP3A11 and yet promote the expression of the hepatoblast markeralbumin. The addition of either FGF10 or HGF to media containing onlyEGF (without R-spondin1 and without nicotinamide), facilitated theexpression of the mature marker CYP3A11, albeit at very low levels (FIG.8E). To identify additional compounds that might facilitate hepatocytedifferentiation, we used two different approaches, both based upon baseconditions of: EGF+HGF and/or FGF10.

The first approach involved testing a series of compounds in addition tothe EGF+FGF10 or HGF condition. A complete list of the compoundsanalysed is shown in table 2.

TABLE 2 Result Compounds Signal Concentration Alb CYP3AII Exendin4Glucagon like Sigma 0.1-1 uM peptide 2 analog E7144 Retinoic AcidRAR-RXR Sigma 25 nM receptor ligand Retinoic Acid + Exendin 4 SonicHedgehog Invitrogen 500-100 ng/ml C25II BMP4 BMP signaling Peprotech 20ng/ml 120-05 DAPT Gamma-secretase Sigma 10 nM inhibitor D5942 A8301Alk5/4/7 inhibitor Tocris 50 nM Bioscience 2939 DAPT + A8301 +++ +++FGF4 FGFR1,2 ligand Peprotech 50 ng/ml FGF1 FGFR1,2,3,4 Peprotech 100ng/ml ligand 450-33A Dexamethasone Sigma 10 μM-1 mM D4902 25MGOncostatin M R&D 10-1000 ng/ml (OSM) systems VEGF + DEXA 495-MO- 025FGF4 + OSM + Dexa IGF peprotech 100 ng/ml Valproic acid histone Stemgent250 μM deacetylase 04-0007 inhibitor and regulator of ERK, PKC Wnt/β-catenin pathways Sodium Butyrate histone Stemgent 250 μM deacetylase04-0005 inhibitor BIX01294 G9a HMTase Stemgent 1 μM inhibitor 04-0002 RG108 DNA Stemgent 1 μM methyltransferase 04-0001 inhibitor TSA 100 nM + −Hydrocortisone glucocorticoid Sigma 5 nM H6909 Oncostatin M R&D 10-1000ng/ml (OSM) systems 495-MO- 025 ARA Sigma A 500 nM 0937 R 59022Diacylglycerol Sigma D 500 nM-50 nM + + kinase inhibitor 5919 Arterenolbitrartre: andrenoreceptor sigma 500 nM-50 nM- — agonist A 0937 5 nM LIF10³ PD 035901 MEK1 inhibitor Axon 500 nM Medchem cat n 1386 CHIR99021GSK3 inhibitor Axon 3 uM Medchem cat n 1408 DMSO 1% L-Ascobic acid Sigma1 mM 077K13021 VEGF Peprotech Matrigel 50% Matrigel 20%

The second approach took into account knowledge from publisheddevelopmental studies regarding the expression of the transcriptionfactors essential to achieve biliary and hepatocyte differentiation invivo. A comparative analysis of the expression of transcription factorsin the organoids under E or ER or ENRW conditions supplemented withFGF10, HGF and Nicotinamide is shown in FIG. 8. All the transcriptionfactors required for Hepatocyte specification were present, besides tbx3and prox1. However, we also noticed that the expression of specificbiliary transcription factors was highly upregulated in the culturescontaining R-spondin1 (R), indicating that the culture gene expressionwas unbalanced towards a more biliary cell fate.

Notch and TGF-beta signaling pathways have been implicated in biliarycell fate in vivo. In fact, deletion of Rbpj (essential to achieveactive Notch signalling) results in abnormal tubulogenesis (Zong Y.Development 2009) and the addition of TGF-beta to liver explantsfacilitates the biliary differentiation in vitro (Clotman F. Genes andDevelopment 2005). Since both Notch and TGF-beta signalling pathwayswere highly upregulated in the liver cultures (FIG. 9) we reasoned thatinhibition of biliary duct cell-fate might trigger the differentiationof the cells towards a more hepatocytic phenotype. A8301 was selected asan inhibitor of TGF-beta receptor ALK5, 4, and 7 and DAPT as inhibitorof the gamma-secretase, the active protease essential to activate theNotch pathway. We first cultured the cells for 2 days in the expansionconditions (ER media) and at day 2 (FIG. 10A) we started thedifferentiation conditions by adding the combination of the differentcompounds. Media was changed every other day, and the expression ofdifferentiated markers was analysed 8-9 days later. The ER and ENRWconditions were used as negative control.

The combination of EGF+FGF10 with DAPT and A8301 resulted insurprisingly large enhancement of expression of the hepatocyte markersanalysed (CYP3A11, TAT, Albumin) (FIG. 10B). The effect was alreadydetectable by day 5 and peaked at days 8-9 (FIG. 10C). The maximalconcentration efficiency was achieved at 10 uM (DAPT) and 50 nM (A8301)(FIG. 10D) respectively. The addition of dexamethasone (a knownhepatocyte differentiation molecule) did not result in any improvementin gene expression. The combination of EGF, FGF10, A8301 and DAPT notonly enhances the expression but also increases the number ofhepatocyte-like cells, as assessed by immunofluorescent against thehepatocyte markers albumin and 2F8, and Xgal staining on AlbCreLacZderived organoids (FIGS. 10E & F). Therefore, we can conclude that theaforementioned differentiation protocol facilitates the generation ofhepatocyte-like cells in vitro from liver stem cell cultures.

Example 3—Human Liver Organoids

Using these expansion conditions (ERFHNic and ENRWFHNic) we have alsobeen able to expand human biliary-derived cultures (FIG. 11) with theaddition of 500 uM TGF beta inhibitor (A83-01) to the expansion medium.

Material and Methods (for Examples 1-3)

Liver Culture-Biliary Duct Isolation

Isolated adult liver tissue was washed in cold Advanced-DMEM/F12(Invitrogen) and then, the tissue was chopped into pieces of around 5 mmanimals and further washed with cold dissociation buffer (collagenase,dispase, FBS in DMEM media). The tissue fragments were incubated withthe dissociation buffer for 2 h at 37° C. Then, the tissue fragmentswere vigorously suspended in 10 ml of cold isolation buffer with a 10 mlpipette. The first supernatant containing death cells was discarded andthe sediment was suspended with 10-15 ml of dissociation buffer. Afterfurther vigorous suspension of the tissue fragments the supernatant isenriched in biliary ducts. This procedure is repeated until enoughbiliary ducts are obtained.

Isolated biliary ducts are pelleted and mixed with 50 μl of MATRIGEL™(BD Bioscience), seeded on 24-well tissue culture plates and incubatedfor 5-10 min at 37° C. until complete polymerization of the MATRIGEL™.After polymerization, 500 μl of tissue culture media are overloaded.

Media Composition:

Advanced-DMEM/F12 supplemented with B27, N2, 200 ng/ml N-Acetylcysteine,50 ng/ml EGF, 1 μg/ml R-spondin1, gastrin: 10 nM, FGF10 100 ng/ml,Nicotinamide 10 mM and HGF: 50 ng/ml and 50% Wnt conditioned media.

The entire medium was changed every 2 days. After 1 week, Wntconditioned media is withdrawal and the formed organoids removed fromthe MATRIGEL™ using a 1000 μl pipette and were dissociated mechanicallyinto small fragments and transferred to fresh MATRIGEL™. Passage wasperformed in 1:4 split ratio once or twice per week. Under theseconditions cultures have been maintained for at least 6 month.

Reagents

Human Hepatocyte Growth Factor (HGF) was purchased from Peprotech, EGFinvitrogen, R-Spondin Nuvelo, Noggin peprotech, FGF10 Peprotech, gastrinSigma Aldrich, nicotinamide Sigma.

Microarray

For the expression analysis of Lgr5-derived liver cultures, RNA wasisolated using a Qiagen RNAase kit, from adult liver or from livercultures cultured in media without Wntcm and Noggin (ER) or with Wntcmand Noggin (ENRW). 150 ng of total RNA was labelled with low RNA InputLinear Amp kit (Agilent Technologies, Palo Alto, Calif.). Universalmouse Reference RNA (Agilent) was differentially labelled and hybridizedto either adult liver tissue or ER or ENRW treated cultures. A 4×44KAgilent Whole Mouse Genome dual colour Microarrays (G4122F) was used.Labelling, hybridization, and washing were performed according toAgilent guidelines.

Example 4—Lgr5 Expression is Upregulated Following Liver Injury

In the liver, Wnt signalling is active in central vein areas. We haverecently observed that Wnt signaling plays a key role in livermetabolism (Boj et al. personal communication). In the liver duct cells,Wnt signalling is activated following liver injury (Hu et al 2007,Gastroenterology, 133(5): 1579-91). Similarly, using the Axing-LacZallele, which represents a faithful, general reporter for Wntsignalling, we also have observed upregulation of Wnt signaling in thewhole liver parenquima after injection of the Wnt agonist Rspo1 (seeFIG. 12A) or following liver injury by the hepatotoxic compound carbontetrachloride (CC14) (see FIG. 12B).

The Wnt target gene Lgr5 marks stem cells in several activelyself-renewing tissues, but has not previously been reported to beexpressed upon injury. Our previously described Lgr5-LacZ knockin mice(Barker et al, 2007, Nature 449 (7165): 1003-7) show that Lgr5 isessentially undetectable in healthy liver although a residual mRNAexpression is detected by qPCR. Following injection of CC14 on Lgr5-LacZknockin mice (see Barker et al, 2007, supra for LacZ mice and Furuyama Ket al., Nat Genetics, 43, 34-41, 2001 for description of CC14 method),we observed a clear expression of the reporter in newly formed budstructures in the liver (see FIG. 13A), peaking at day 6.5 after injuryand being maintained up to day 9 to show a clear decay once the liver iscompletely regenerated at day 13 after injury (see FIG. 13A, top rightpanel). No expression of the reporter was detected in wild-typelittermates undergoing similar injury protocol (see FIG. 13A, bottomright panel).

The appearance of Lgr5 expression at sites of active regeneration,suggested that Lgr5 might herald de novo activation by Wnt ofregenerative stem cells/progenitors upon injury. Indeed, we found thatthe novo appearing Lgr5 cells do not express markers of mature livercells (K19 or FAH) or stellate cells (SMA) but instead, they arepositive for the recently described liver progenitor marker Sox9 (FIG.13B). This means that Lgr5+ cells, which are the starting point forobtaining in vitro organoids, can be obtained from liver fragments byinducing liver injury or by stimulating Wnt signalling with R-spondin.The induction of Lgr5 expression in liver cells by injury or byR-spondin may be carried out in vivo before the cells are obtained, exvivo in an isolated liver, or in vitro in a liver fragment or populationof liver cells.

Example 5—Long-Term Expansion of Liver Organoid Cultures

In example 1, it was found that the combination of EGF (50 ng/ml) andR-spondin 1 (1 ug/ml) supplemented with FGF10 (100 ng/ml), HGF (25-50ng/ml) and Nicotinamide (1-10 mM), were preferable for the long termmaintenance of the cultures. We now also have evidence that the threesupplemental factors (FGF10, HGF and Nicotinamide) added to EGF andR-spondin1 are all necessary for the expansion of the cultures forlonger than 3 months. To assess that, we isolated biliary ducts from theliver parenquima, as shown in FIG. 14 (K19 staining was used to confirmthe identity of the isolated structures), and generated liver organoidcultures by culturing them with: i) EGF; or ii) EGF and R-spondin1 plusFGF10 or HGF or Nicotinamide; or iii) EGF and R-spondin1 plus FGF10 andHGF and Nicotinamide (ERFHNic). We have split the cultures once a weekfor a total period of 14 weeks. Results confirmed, as reported inexamples 1 and 2, that EGF, R-spondin1 and Nicotinamide combined withFGF10 are essential for the growth and self-renewal of the livercultures. After 10 passages, the cultures lacking HGF showed a growthdisadvantage compared to the cultures supplemented with HGF. Althoughstill viable, the proliferation ratio decreased to 1:2-1:4 compared tothe 1:6-1:8 of the cultures supplemented with the complete combination(FGF10, HGF, and Nicotinamide). After 15 passages, the cultures withERFNic not supplemented with HGF were no longer viable. Therefore, theseresults suggest that HGF is essential for maintaining a goodproliferating rate after long-term maintenance (FIG. 15).

Example 6—Markers Expressed in Liver Organoids Under DifferentiationConditions

Using the differentiation protocol described in example 2, we were ableto detect a hepatoblast marker (albumin) and a hepatocyte surface markerin the liver organoids. To quantify the number of these hepatocyte-likecells, we performed flow cytometry analysis of the cultures using ahepatocyte surface marker. We observed that, whereas in the expansionculture condition almost no hepatocyte surface marker-positive cellswere detected, after differentiation, up to 35% of the cells werepositive for this hepatocyte surface marker (see FIGS. 17B&C).

We then analysed the gene expression profile of the mouse liverorganoids under these differentiation conditions (FIG. 16 and FIG. 1, wesee strong upregulation of eg Alb, FAH, and TAT and the Cyp3 genes). Wefound that the gene expression of the mouse liver organoids afterdifferentiation resemble that of mature mouse hepatocytes and/or mouseliver.

Example 7—Transplantation of Liver Organoids into Mice

Cells were taken from the organoids that had been grown using ERFHNicexpansion conditions and EAFD differentiation conditions and weretransplanted into immunodeficient strain of mice deficient in thetyrosine catabolic enzyme fumarylacetoacetate hydrolase (FAH), a mousemodel for Tyrosinemia type I human disease (Azuma et al. 2007, NatureBiotech, 25(8), 903-910). The transplantation schedule is shown in FIG.17D. Preliminary results show that scattered FAH positive cells can befound in the liver parenquima of the FAH deficient mice, indicating thatliver cells derived from the organoid cultures have engrafted into therecipient livers (see FIG. 17A, right-hand side). Furthermore,significantly increased numbers of K19 positive cells were also detectedin the livers of the recipient mice. This suggests that theorganoid-derived transplanted cells are able to generate both lineagesin vivo: hepatocytes (as demonstrated by the FAH marker) andcholangyocytes (as demonstrated by the K19 marker) (see FIG. 17A, lefttop panel). This was further supported by flow cytometry analysis oftransplanted cells that had come from two separate clones from twoseparate cultures (FIGS. 17B and 17C respectively). The Lgr5+ cells weretransduced with a virus containing GFP and flow cytometry analysis wascarried out after differentiation. Cells that were positive for thehepatocyte surface marker show a larger scatter indicating larger cells,which represent granularity and maturity i.e. mature hepatocyte cells.The cells that were negative for the hepatocyte surface marker resultedin less scattering indicating smaller cells i.e. less matureprogenitors. Therefore, all cell types are present (mature and immaturecells) in a differentiating culture. The rest of the differentiatedcells, so the cells not used for FACS analysis were used for thetransplantation experiments.

Example 8

Organoids from mouse liver cultured in accordance with a method of theinvention were analysed using microarray analysis to determine whichgenes are expressed and which genes are not expressed.

Example 9

Organoids from human liver cultured using the EM1, EM2 and DM media ofthe invention and human liver were analysed using oligonucleotidemicroarray analysis to determine which genes are expressed and whichgenes are not expressed. A significantly different gene expressionprofile was noticeable between the genes expressed in expansion media,the genes expressed in differentiation medium and the genes expressed inadult liver. The trend for hepatocyte gene expression is roughly thesame as for in the mouse but the differentiation of the organoids wasless than in the mouse liver organoids. This may be due to use of thehuman cell used.

As often happens in an analysis using an oligonucleotide microarray,Lgr5 and Tnfrsf19 were not detected. However, they were found to bepresent in organoids cultured in the expansion medium.

Materials & Methods (for Examples 4 to 7)

Animal Treatment

Two-Eight month old Lgr5LacZ or Axin2-LacZ or WT littermates BL6/BalbcF1 mice received an intraperitoneal injection of 0.8 ml/kg of CCL4disolved in corn oil (n=) or corn oil alone (n=). Mice were sacrificed 2or 5 or 9 or 13 days later and the liver was isolated and furtherprocessed for RNA or bgalactosidase staining.

β-Galactosidase (lacZ) Staining

Liver tissues were isolated and immediately incubated for 2 hours in a20-fold volume of ice-cold fixative (1% Formaldehyde; 0.2%Gluteraldehyde; 0.02% NP40 in PBS0) at 4° C. on a rolling platform. Thefixative was removed and the tissues washed twice in washing buffer(PBS0; 2 mM MgCl₂; 0.02% NP40; 0.1% NaDeoxycholate) for 20 minutes atroom temperature on a rolling platform. The β-galactosidase substrate (5mM K₃FE(CN)₆; 5 mM K₄Fe(CN)₆.3H₂O; 2 mM MgCl₂; 0.02% NP40; 0.1%NaDeoxycholate; 1 mg/ml X-gal in PBS0) was then added and the tissuesincubated in the dark at 37° C. for 2 h and overnight at roomtemperature. The substrate was removed and the tissues washed twice PBS0for 20 minutes at room temperature on a rolling platform. The tissueswere then fixed overnight in a 20-fold volume of 4% Paraformaldehyde(PFA) in PBS0 at 4° C. in the dark on a rolling platform. The PFA wasremoved and the tissues washed twice in PBS0 for 20 minutes at roomtemperature on a rolling platform.

The stained tissues were transferred to tissue cassettes and paraffinblocks prepared using standard methods. Tissue sections (4 μM) wereprepared and counterstained with neutral red.

R-Spondin1 Treatment

Axin2-lacZ mice aged 6-8 weeks were injected IP with 100 μg of purifiedhuman R-spondin1 and sacrificed 48 hours later for LacZ expressionanalysis in the liver.

RT-PCR

RNA was extracted from gastric cell cultures or freshly isolated tissueusing the RNeasy Mini RNA Extraction Kit (Qiagen) andreverse-transcribed using Moloney Murine Leukemia Virus reversetranscriptase (Promega). cDNA was amplified in a thermal cycler (GeneAmpPCR System 9700; Applied Biosystems, London, UK) as previously described(Huch et al., 2009). Primers used (SEQ ID NOs:1-26 from top to bottom,respectively) are shown in Table 3 below.

TABLE 3  Primers for RT-PCR PCR Gene product Gene name Symbol Sequence(bp) cytochrome P450, family 3, CYP3A11 fw TGGTCAAACGCCTCTCCTTGCTG 100subfamily a, polypeptide 11 rv ACTGGGCCAAAATCCCGCCG Glucose-6-phoshataseG6P fw GAATTACCAAGACTCCAGG 581 rv TGAGACAATACTTCCGGAGG Keratin 19 Krt19fw GTCCTACAGATTGACAATGC 549 rv CACGCTCTGGATCTGTGACA Albumin Alb fwGCGCAGATGACAGGGCGGAA 358 rv GTGCCGTAGCATGCGGGAGG t-box 3 Tbx3 fwAGCGATCACGCAACGTGGCA 441 rv GGCTTCGCTGGGACACAGATCTTTProspero-related-homeobox Prox1 fw TTCAACAGATGCATTACC 270 protein 1 rvTCTTTGCCCGCGATGATG Fumarylacetoacetate- Fah fw ACGACTGGAGCGCACGAGAC 183hydrolase rv AGGGCTGGCTGTGGCAGAGA Tyrosine aminotransferase Tat fwTTTGGCAGTGGCTGAAAGGCA 258 rv GGGCCCAGGATCCGCTGACTTryptophan2,3-dioxygenase Tdo2 fw ACTCCCCGTAGAAGGCAGCGA 583 rvTCTTTCCAGCCATGCCTCCACT Leucine-rich repeat- Lgr5 fwGGAAATGCTTTGACACACATTC 413 containing G-protein rvGGAAGTCATCAAGGTTATTATAA coupled receptor 5 Transthyretin TTR fwATGGTCAAAGTCCTGGATGC 220 rv AATTCATGGAACGGGGAAAT Glucokinase Gck fwAAGATCATTGGCGGAAAG 193 rv GAGTGCTCAGGATGTTAAG hypoxanthine Hprt fwAAGCTTGCTGGTGAAAAGGA 186 phosphoribosyltransferase rvTTGCGCTCATCTTAGGCTTTImmunohistochemistry

Immunostaining procedure used here was previously described in Huch etal. 2009. Briefly, five-micrometer sections were deparaffinized,rehydrated, and tissue sections were permeabilized using PBS-T (PBS;Tween20 0.1%). When required, sections were treated with 10 mM citratebuffer (pH 6.0) for antigen retrieval, blocked using Universal blockingbuffer (BioGenex)) and incubated with the primary antibody. Then,sections were washed twice with PBS and incubated with peroxidaseconjugated secondary antibodies. DAB+(DAKO) was used as a chromogensubstrate. Sections were counterstained with Mayer's hematoxylin andvisualized on a Leica DMR microscope. The primary antibodies used wererabbit anti-Sox9 (1:600; 1 h at RT, Millipore), mouse anti-SMA (1:1000,overnight at 4° C., Sigma), rabbit anti-FAH (1:5000; overnight 37° C.,gift from M. Grompe), rabbit anti-K19 (1:500; overnight 4° C., gift fromM. Grompe). The peroxidase conjugated secondary antibodies used wereMouse or Rabbit Brightvision (Immunologic).

Immunofluorescence

For whole mount staining, organoids or isolated biliary ducts were fixedwith acetone (organoids) or PFA4% (biliary ducts) for 30 min, washedonce with PBS, permeabilized with PBS 0.3% Triton-X100 for 5 min,blocked using Universal blocking solution (Power block HK085-5KEBioGenex) and incubated overnight with the primary antibodies diluted inPBS1% FBS. Following several washes in PBS, samples were incubated withthe secondary antibody. Nuclei were stained with Hoescht33342. Imageswere acquired using confocal microscopy (Leica, SP5). Three-dimensionalreconstruction was performed using Volocity Software (Improvision). Theprimary antibodies used were rabbit anti-K19 (1:500; gift from M.Grompe), rat anti-hepatocyte surface marker (1:50, gift M. Grompe), goatanti-albumin (1:50, santa Cruz). The secondary antibodies used were allraised in donkey and conjugated to different Alexa fluorofores (donkeyanti-goat 568, donkey anti rat-488, donkey anti rabbit-647, Molecularprobes).

Flow Cytometry

Dissociated cells were resuspended at 1×10⁴ cells per milliliter in 1 mlof DMEM+2% FBS prior to the addition of MIC1-1C3 hybridoma supernatantat a 1:20 dilution or OC2-2F8 hybridoma supernatant at a 1:50 dilution,and incubated for 30 min at 4° C. After a wash with cold Dulbecco'sPhosphate Buffered Saline (DPBS), cells were resuspended in DMEM+2% FBScontaining a 1:200 dilution of APC-conjugated goat anti-rat secondaryantibody adsorbed against mouse serum proteins (Jackson Immunoresearch).Propidium iodide staining was used to label dead cells for exclusion.Cells were analyzed and sorted with a Cytopeia in FluxV-GS(Becton-Dickenson).

Transplantation Assay

The injection of sorted cell populations to the spleen and thewithdrawal of NTBC to induce hepatocyte selection were performed asdescribed previously (Overturf et al. 1996). Drug withdrawal was done inperiods of 3 wk, followed by readministration until normal weight wasrestored in the recipient animals.

The invention claimed is:
 1. A method for obtaining a liver organoidcomprising Lgr5+ epithelial stem cells, wherein said method comprises:culturing and expanding Lgr5+ epithelial stem cells from a liverfragment or a liver biliary duct in contact with an extracellular matrixin the presence of a medium, the medium comprising a basal medium foranimal or human cells to which is added: a Wnt agonist, EGF, an FGF andNicotinamide in amounts sufficient to form a liver organoid comprisingLgr5+ epithelial stem cells which are capable of at least 5 passages invitro.
 2. The method according to claim 1, wherein after at least oneday, the medium is changed to a second medium.
 3. The method accordingto claim 2, wherein the second medium is a second expansion mediumcomprising a basal medium to which is added: EGF, an FGF, an HGF,Nicotinamide and an Rspondin.
 4. The method according to claim 2,wherein the second medium is a differentiation medium comprising a basalmedium for animal or human cells to which is added EGF, FGF and/or HGF,and a Notch inhibitor.
 5. The method according to claim 2, wherein themethod comprises culturing said epithelial stem cells in a cell culturemedium comprising or consisting of a basal medium for animal or humancells to which is added EGF, a BMP inhibitor, R-spondin and Wnt; andsubsequently in a second expansion medium which comprises or consists ofa basal medium for animal or human cells to which is added: EGF, an FGF,and HGF, Nicotinamide, and an R-spondin; and subsequently in adifferentiation medium which comprises or consists of a basal medium foranimal or human cells to which is added: EGF, FGF and/or HGF, a TGF-betainhibitor, and a Notch inhibitor.
 6. The method according to claim 1,wherein the Wnt agonist is selected from the group consisting of: a Wntfamily member, R-spondin 1, R-spondin 2, R-spondin 3, R-spondin 4,Norrin and a GSK-inhibitor.
 7. The method according to claim 1, whereinone or more of a BMP inhibitor, a TGF-beta inhibitor and an HGF areadded to the medium.
 8. The method according to claim 7, wherein aTGF-beta inhibitor is added to the medium and wherein the TGF-betainhibitor is a small molecule inhibitor selected from the groupconsisting of: A83-01, SB-431542, SB-505124, SB-525334, LY 364947,SD-208 and SJN
 2511. 9. The method according to claim 1, wherein theculture medium further comprises HGF.
 10. A method for obtaining a liverorganoid comprising Lgr5+ epithelial stem cells, wherein said methodcomprises: culturing and expanding Lgr5+ epithelial stem cells from aliver fragment or a liver biliary duct in contact with an extracellularmatrix in the presence of a medium, the medium comprising a basal mediumfor animal or human cells to which is added: a Wnt agonist, EGF, an HGFand Nicotinamide in amounts sufficient to form a liver organoidcomprising Lgr5+ epithelial stem cells, which are capable of at least 5passages in vitro.
 11. The method according to claim 10, wherein theculture medium further comprises FGF.